Two-step template switching reverse transcription methods

Abstract

Methods are described for enhancing complementary DNA (cDNA) synthesis from a ribonucleic acid (RNA) template during reverse transcription by an enzyme having template switching activity. The methods provide for enhancing the processivity of a reverse transcriptase.

Description

TWO-STEP TEMPLATE SWITCHING REVERSE TRANSCRIPTION METHODSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 572,576, filed April 1, 2024, which is hereby incorporated by reference in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under HG011868 awarded by National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an XML Document formatted sequence listing with a file name “047162-5372- 00WO Sequence Listing.xml,” having a creation date of April 1, 2025, and having a size of 22,399 bytes. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.BACKGROUND

[0004] Polymerase enzymes, such as reverse transcriptases, are useful in a variety of commercial settings for synthesizing complementary deoxyribonucleic acids (cDNAs) from ribonucleic acid (RNA) templates. For example, polymerases are used in methods of assessing gene expression, such as RNA sequencing or reverse transcription coupled with quantitative polymerase chain reaction (qRT-PCR). While polymerases (e.g., reverse transcriptases) are critical to these methods, accurate measurement of gene expression is limited by the ability of the reverse transcriptase to synthesize full-length complementary DNAs (cDNAs) (i.e., cDNA complementary to an entire target RNA from its 5’ terminus to its 3’ terminus). Thus, there is a need in the art to improve the length of cDNAs synthesized by polymerases through reverse transcription and template switching.SUMMARY

[0005] The present disclosure features two-step procedures for using template-switching reverse transcriptases, and related compositions thereof. The methods described herein are useful for enhancing the yield and efficiency of cDNA synthesis in these reactions, as well as reducing fragmentation product production. In an embodiment, the methods described herein also provide a dissection of the reverse transcription and template switching activity of exemplary enzymes, wherein the reverse transcription reaction is performed prior to the template switching reaction. In another aspect, the present disclosure comprises a method of enhancing complementary DNA (cDNA) synthesis, e.g., from an RNA template, by a polymerase having reverse transcriptase and template switching activity, e.g., a reverse transcriptase. In some embodiments, the method comprises preparing a first reaction mixture comprising: (i) a target RNA, e.g., a plurality of target RNAs; (ii) a reverse transcription primer; (iii) a deoxyribonucleotide triphosphate (dNTP) solution; and (iv) a polymerase, e.g., a reverse transcriptase. In some embodiments, the method comprises allowing the first reaction mixture to incubate for a time period. In some embodiments, the method comprises preparing a second reaction mixture, comprising: (v) the first reaction mixture after incubating for a time period; and (vi) a template switching oligonucleotide. In some embodiments, the method enhances synthesis of cDNA. In some embodiments, the second reaction mixture further comprises: (vii) a deoxyadenosine triphosphate (dATP) solution; (viii) a polyethylene glycol; and (ix) a polymerase, e.g., a reverse transcriptase.

[0006] The two-step method comprises a reaction mixture comprising (i) a target RNA, e.g., a plurality of target RNAs; (ii) a reverse transcription primer; (iii) a deoxyribonucleotide triphosphate (dNTP) solution; and (iv) a polymerase, e.g., a reverse transcriptase. In some embodiments, the level of the target RNA in the mixture is between about 1 pg to about 0.1 pg. In some embodiments, the reverse transcription primer is between 5 and 500 nucleotides in length. In some embodiments, the reverse transcription primer is complementary to the 3’ end of the target RNA. In some embodiments, mixture further comprises a plurality of input RNA sequences (e.g., non-target RNA). In some embodiments, the polymerase is a DNA polymerase or an RNA polymerase. For example, the polymerase may be a reverse transcriptase. In some embodiments, the reverse transcriptase is derived from a virus, an intron, a telomerase, a retrotransposon, a polymerase with reverse transcriptase activity, or an engineered polymerasewith reverse transcriptase activity. In some embodiments, the reverse transcriptase is a group II intron reverse transcriptase, a telomerase reverse transcriptase, a viral reverse transcriptase or a retroviral reverse transcriptase. In some embodiments, the reverse transcriptase comprises MarathonRT™, UltraMarathonRT®, Induro®, Maxima™ H Minus, SuperScript™ II, SuperScript™ III, SuperScript™ IV, PrimeScript™, Transcriptor, GoScript™, ProtoScript® II, SMARTScribe™, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, Bombyx Mori RT, telomerase RT, TGIRT™, or a fragment, variant, mutant, or derivative thereof. In some embodiments, the reverse transcriptase comprises MarathonRT™ or UltraMarathonRT®. In some embodiments, the reverse transcriptase comprises UltraMarathonRT®. In some embodiments, the reverse transcriptase comprises MarathonRT™.

[0007] In another aspect, the present disclosure comprises methods of enhancing cDNA synthesis. In some embodiments, the enhancing of cDNA synthesis comprises increasing the length of the cDNA synthesized by the polymerase, e.g., increasing the length of the cDNA by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more. For example, the cDNA synthesized may be full-length cDNA, e.g., cDNA that is complementary to an entire target RNA sequence, e.g., from the 5’ terminus of the target RNA to the 3 ’ terminus of the target RNA. In some embodiments, the cDNA is between about 10 nt and 100,000 nt in length, e g., about 400 nt, 800 nt, 1,000 nt, 1,500 nt, 2,000 nt, 2,500 nt, 3,000 nt, 3,500 nt, 4,000 nt, 4,500 nt, 5,000 nt, 5,500 nt, 6,000 nt, 6,500 nt, or 7,000 nt, 8,000 nt, 9,000 nt, 10,000 nt, 15,000 nt, 20,000 nt, 30,000 nt, 40,000 nt, 50,000 nt, 60,000 nt, 70,000 nt, 80,000 nt, 90,000 nt, or 100,000 nt or greater in length. In some embodiments, the average cDNA length is between about 1,500 nt and 10,000 nt in length, e.g., about 1,500 nt, 2,000 nt, or 2,500 nt, 3,000 nt, 3,500 nt, 4,000 nt, 4,500 nt, 5,000 nt, 6,000 nt, 7,000 nt, 8,000 nt, 9,000 nt, or 10,000 nt or greater in length. In some embodiments, the cDNA is synthesized from total cellular RNA to generate a cDNA library.

[0008] The two-step method for reverse transcription and template switching of the present disclosure may allow for each of the reverse transcription and template switching reactions to be carried out under different conditions to enhance cDNA synthesis. In some embodiments, the reverse transcription step and the template switching step are performed under the same conditions, e.g., the same enzyme concentration, temperature, incubation time, orbuffer. In some embodiments, the reverse transcription step and the template switching step are performed under different conditions, e.g., different enzyme concentration, temperatures, incubation times, or buffers. In some embodiments, the enhancing of cDNA synthesis comprises increasing the yield of the cDNA synthesized by the polymerase, e.g., increasing the length of the cDNA by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more.

[0009] The two-step method comprises a first reverse transcription step. In some embodiments, the reverse transcription reaction in the first step is incubated for at least 10 minutes before being contacted with the template switching reaction components in the second step, e.g., incubated for at least 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, or 150 minutes or more.

[0010] In yet another aspect, the present disclosure comprises a method comprising: (i) detecting the level, identity or concentration of a target RNA; (ii) increasing the signal to noise ratio of a target RNA; and / or (iii) increasing the processivity of the reverse transcriptase reaction, compared to a reference standard.

[0011] In another aspect, the present disclosure comprises a kit for enhancing complementary DNA (cDNA) synthesis, e.g., from an RNA template, comprising: (i) a polymerase having reverse transcriptase and template switching activity (e.g., a reverse transcriptase, e.g., MarathonRT™); (ii) a deoxyribonucleotide triphosphate (dNTP) solution; (iii) a buffer solution; and (iv) a template switching oligonucleotide. In some embodiments, the kit further comprises a reverse transcription primer. In some embodiments, the kit further comprises a deoxyadenosine triphosphate (dATP) solution. In some embodiments, (iv) is provided separately from (i)-(iii). In some embodiments, the dATP is provided together with the template switching oligonucleotide. In some embodiments, the polymerase comprises MarathonRT™, UltraMarathonRT®, Induro®, Maxima™ H minus, SuperScript™ II, SuperScript™ III, SuperScript™ IV, PrimeScripf™, Transcriptor, GoScript™, ProtoScript® II, SMARTScribe™, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, TGIRT™, or a fragment, variant, mutant, or derivative thereof. In some embodiments, the polymerase comprises a group II intron RT, e g., UltraMarathonRT®. In some embodiments, the kit comprises MarathonRT™ or UltraMarathonRT®. In someembodiments, the template switching oligonucleotide is between 5 and 500 nucleotides in length. In some embodiments, the reverse transcription primer is between 5 and 500 nucleotides in length.

[0012] In some embodiments, the kit is useful for: (i) acquiring a value for the presence of a target ribonucleic acid (RNA) in a mixture; (ii) detecting the level, identity or concentration of a target RNA; (iii) increasing the signal to noise ratio of a target RNA; (iv) preparing a library for the target RNA templates; and / or (v) increasing the processivity of the reverse transcriptase reaction.BRIEF DESCRIPTION OF DRAWINGS

[0013] FIG. 1 is a schematic representation of a one-step reverse transcription and template switching procedure, e.g., reverse transcription and template switching reactions performed concurrently. “RT” indicates reverse transcription; “TS” indicates template switching; “dATP” indicates deoxyadenosine triphosphate; “dNTPs” indicate deoxyribonucleotide triphosphates, e.g., deoxyadenosine triphosphate, deoxythymidine triphosphate, deoxycytidine triphosphate, or deoxyguanosine triphosphate; “MRT” indicates MarathonRT™ reverse transcriptase; “TSO” indicates template switching oligonucleotide.

[0014] FIG. 2 is a schematic representation of a two-step reverse transcription and template switching procedure, e.g., reverse transcription and template switching reactions performed separately. “RT” indicates reverse transcription; “TS” indicates template switching; “dATP” indicates deoxyadenosine triphosphate; “dNTPs” indicate deoxyribonucleotide triphosphates, e.g., deoxyadenosine triphosphate, deoxythymidine triphosphate, deoxycytidine triphosphate, or deoxyguanosine triphosphate; “MRT” indicates MarathonRT™ reverse transcriptase; “TSO” indicates template switching oligonucleotide.

[0015] FIG. 3A is an image of a bioanalyzer electrophoresis profde of the PCR- amplified cDNA library resulting from one-step, e.g., concurrent, reverse transcription and template switching reactions from total cellular RNA input. FIG. 3B is an image of a bioanalyzer electrophoresis profde of the PCR-amplified cDNA library resulting from two-step, e.g., separate, reverse transcription and template switching reactions from total cellular RNA input.

[0016] FIG. 4 is an image of a bioanalyzer electrophoresis profde of the PCR-amplified cDNA library resulting from two-step, e.g., separate, reverse transcription and templateswitching reactions from total cellular RNA input. The temperature and incubation time for each of the reverse transcription (RT) and template switching (TS) steps was varied.

[0017] FIG. 5 is an image of a bioanalyzer electrophoresis profile of the PCR-amplified cDNA library resulting from two-step, e.g., separate, reverse transcription and template switching reactions from total cellular RNA input. The amount of MarathonRT™ enzyme added at each of the reverse transcription (RT) and template switching (TS) steps was varied.

[0018] FIG. 6 is an image of a bioanalyzer electrophoresis profile of the PCR-amplified cDNA library resulting from two-step, e.g., separate, reverse transcription and template switching reactions from total cellular RNA input. The buffer used in each of the reverse transcription (RT) and template switching (TS) steps was varied.DESCRIPTION

[0019] The present disclosure features methods for enhancing complementary DNA (cDNA) synthesis from a ribonucleic acid (RNA) template, e.g., present in a mixture, during reverse transcription by an enzyme, e.g., a reverse transcriptase (RT), having template switching activity. In an embodiment, the improving comprises separating the reverse transcription and template switching reactions into individual steps. In an embodiment, the enhancing comprises increasing the length of cDNA synthesized by a reverse transcriptase. In other embodiments, the methods described herein provide for enhancing the processivity of a reverse transcriptase.Definitions

[0020] So that the disclosure may be more readily understood, certain technical and scientific terms used herein are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0021] As used herein, including the appended claims, the singular forms of words such as "a," "an," and "the," include their corresponding plural references unless the context clearly dictates otherwise.

[0022] “About" or “approximately” means when used herein to modify a numerically defined parameter (e.g., yield of cDNA resulting from reverse transcription by a reverse transcriptase), means that the recited numerical value is within an acceptable functional range forthe defined parameter as determined by one of ordinary skill in the art, which will depend in part on how the numerical value is measured or determined, e.g., the limitations of the measurement system, including the acceptable error range for that measurement system. For example, “about” can mean a range of 20% above and below the recited numerical value. As a non-limiting example, the concentration of an anionic polymer in a reaction mixture may be about 0.01 pg / pL to about 100 ng / pL. In some embodiments, the term “about” means that the modified parameter may vary by as much as 15%, 10% or 5% above and below the stated numerical value for that parameter. Alternatively, particularly with respect to certain properties of an anionic polymer in a reaction mixture, such as increasing the yield of cDNA produced from low abundance nucleic acids in a sample, the term “about” can mean within an order of magnitude above and below the recited value, e.g., within 5-fold, 4-fold, 3-fold, 2-fold or 1-fold.

[0023] “Acquire” or “acquiring”, as used herein, refer to obtaining possession of a value, e.g., a numerical value, or image, or a physical entity (e.g., a sample), by “directly acquiring” or “indirectly acquiring” the value or physical entity. “Directly acquiring” means performing a process (e.g., performing an analytical method or protocol) to obtain the value or physical entity. “Indirectly acquiring” refers to receiving the value or physical entity from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value). Directly acquiring a value or physical entity includes performing a process that includes a physical change in a physical substance or the use of a machine or device. Examples of directly acquiring a value include obtaining a sample from a human subject. Directly acquiring a value includes performing a process that uses a machine or device, e.g., using a fluorescence microscope to acquire fluorescence microscopy data.

[0024] A “nucleotide,” as that term is used herein, refers to an entity comprising a sugar, typically a pentameric sugar; a nucleobase; and a phosphate linking group. In an embodiment, a nucleotide comprises a naturally occurring, e.g., naturally occurring in a human cell, nucleotide, e.g., an adenine, thymine, guanine, cytosine, or uracil nucleotide. “Nucleotide” may be abbreviated as “nt” herein.

[0025] “Nucleic acid”, as used herein, refers to a polymer comprising a nucleotide linked through phosphodiester bonds. In an embodiment, a nucleic acid comprises at least two, and in some embodiments, at least 10, 100, 1,000, or 10,000 nucleotides. In some embodiments, a nucleic acid comprises ribonucleotides, e g., is a ribonucleic acid (RNA). In some embodiments,a nucleic acid comprises deoxyribonucleotides, e.g., is deoxyribonucleic acid (DNA). “Nucleic acid”, as used herein, is interchangeable with “polynucleotide” or “oligonucleotide”. The length of a nucleic acid is referred to herein as a number of bases, e.g., a nucleic acid comprising 1,000 nucleotides has a length of 1,000 bases or 1 kilobase (kb).

[0026] “Complementary DNA” or “cDNA”, as used herein, refers to the deoxyribonucleic acid (DNA) product synthesized through a reverse transcription reaction, e.g., DNA synthesized by reverse transcription using a ribonucleic acid (RNA) molecule as a substrate.

[0027] “Polypeptide”, as used herein, refers to a polymer comprising amino acid residues linked through peptide bonds and having at least two, and in some embodiments, at least 10, 50, 75, 100, 150 or 200 amino acid residues.

[0028] The term “template” as used herein, with respect to a polynucleotide, refers to a single-stranded polynucleotide substrate for a nucleic acid polymerase, e.g., a reverse transcriptase. For example, a nucleic acid polymerase, e.g., a reverse transcriptase, can synthesize a polynucleotide strand that is complementary to the template strand. In some embodiments, a single-stranded RNA polynucleotide can be a template for a reverse transcriptase. A “template" polynucleotide, as used herein, may also be referred to as a “target” polynucleotide. In some embodiments, a target polynucleotide is RNA. In some embodiments, a target polynucleotide is DNA. The term “product” as used herein, with respect to a polynucleotide, refers to the polynucleotide strand synthesized by a nucleotide polymerase. In some embodiments, the nucleotide polymerase is a reverse transcriptase. In some embodiments, the product polynucleotide is a deoxyribonucleic acid (DNA) polynucleotide synthesized by a reverse transcriptase using a ribonucleic acid (RNA) polynucleotide as a template.

[0029] The term “target” as used herein, with respect to a polynucleotide, refers to a polynucleotide intended for detection by a reverse transcriptase, e.g., a polynucleotide for which a value is acquired using a method of reverse transcription. For example, the oligonucleotide primers in a reverse transcription reaction mixture are complementary to the target polynucleotide, thereby allowing the target polynucleotide to be reverse transcribed. In some embodiments, the target polynucleotide comprises a deoxyribonucleic acid (DNA). In some embodiments, the target polynucleotide comprises a ribonucleic acid (RNA).

[0030] The term “reverse transcription” as used herein, with respect to a subject molecule, e.g., a RNA polynucleotide, refers to synthesis of a deoxyribonucleic acid (DNA), e.g., cDNA, polynucleotide using a ribonucleic acid (RNA) polynucleotide as a template.

[0031] The term “reverse transcription activity” as used herein, refers to the capacity of an enzyme, e.g., a polymerase, to perform reverse transcription, e.g., perform a reverse transcription reaction. For example, an enzyme having reverse transcription activity is capable of synthesizing a DNA polynucleotide from an RNA polynucleotide template.

[0032] The term “reverse transcriptase” as used herein refers a nucleic acid polymerase capable of synthesizing a deoxyribonucleic acid (DNA) polynucleotide from a template ribonucleic acid (RNA) polynucleotide. For example, a reverse transcriptase may synthesize a single-stranded complementary DNA (cDNA) polynucleotide product from a messenger RNA (mRNA) expressed in a cell or subject. In some embodiments, an RT may be a templateswitching RT. In some embodiments, the RT comprises a MarathonRT™ reverse transcriptase, an UltraMarathonRT® reverse transcriptase, a Moloney Murine Luekemia Virus reverse transcriptase, an Avian Myeloblastosis Virus reverse transcriptase, Bombyx mori R2 RNA element reverse transcriptase, a TGIRT™ reverse transcriptase, Induro® reverse transcriptase, Maxima™ H minus reverse transcriptase, SuperScript™ II reverse transcriptase, SuperScript™ III reverse transcriptase, SuperScript™ IV reverse transcriptase, PrimeScript™ reverse transcriptase, Transcriptor reverse transcriptase, GoScript™ reverse transcriptase, ProtoScript® II reverse transcriptase, or SMARTScribe™ reverse transcriptase, as well as variants, fragments, and mutants thereof.

[0033] The term “non-templated nucleotide addition” as used herein refers to the addition of nucleotides to the 3’ end of a product polynucleotide synthesized by an enzyme upon reaching the 5’ terminus of a template polynucleotide, e.g., addition of nucleotides to the product polynucleotide that are not comprised in the template polynucleotide. For example, non- templated nucleotide addition can result in a product polynucleotide that comprises a 3’ end which extends beyond the 5’ end of the template polynucleotide and is non-complementary to the template polynucleotide. Typically, non-templated nucleotide addition results in a 1-3 nucleotide overhang, e.g., 1, 2, or 3 nucleotide overhang, at the 3’ end of the product polynucleotide relative to the template polynucleotide.

[0034] The term “template switching” as used herein refers to the process of a polymerase enzyme switching from a first template polynucleotide to a second template polynucleotide while synthesizing a continuous product polynucleotide. Typically, template switching comprises: (i) non-templated nucleotide addition of nucleotides to the 3’ end of the polynucleotide synthesized by the reverse transcriptase upon reaching the 5’ terminus of the template polynucleotide; (ii) base pairing between a template switching oligonucleotide (TSO) and the nucleotide overhang resulting from non-templated addition; and (iii) continued synthesis of the product polynucleotide by the reverse transcriptase using the TSO as the template polynucleotide.

[0035] The term “template switching activity” as used herein, refers to the capacity of an enzyme, e.g., a polymerase, to perform template switching, e.g., perform a template switching reaction. For example, an enzyme having template switching activity is capable of non-templated nucleotide addition, e.g., continuing polynucleotide synthesis without a template sequence upon reaching the 3’ terminus of the template polynucleotide, and switching template polynucleotides, e.g., continuing polynucleotide synthesis upon base pairing of a template switching oligonucleotide (TSO) to the nucleotides added by non-templated nucleotide addition.

[0036] The term “concatemerization” as used herein refers to the linkage of a plurality of the same polynucleotide sequence in series, e.g., the linkage of a plurality of template switching oligonucleotide (TSO) sequences. In some embodiments, concatemerization of a plurality of a TSO can be a result of repeated cycles of non-templated nucleotide addition by a reverse transcriptase followed by template switching by the reverse transcriptase.

[0037] “Carrier RNA” as used herein refers to a supplemental ribonucleic acid (RNA) molecule that improves the yield of enzymatic reactions on nucleic acids, e.g. cDNA produced during reverse transcription, e.g., an RNA molecule that is present in the reaction mixture but is not a target RNA. Typically, a carrier RNA stabilizes template RNA. Alternatively, carrier RNA may improve reverse transcription activity.

[0038] “Polymerase” or “polymerase enzyme”, as used herein, refers to an enzyme capable of forming phosphodiester linkages between nucleotides, e.g., ribonucleotides or deoxyribonucleotides, in a manner that is directed by a template polynucleotide, e.g., a template ribonucleic acid (RNA) or template deoxyribonucleic acid (DNA), thereby generating a polynucleotide strand that is complementary to the template polynucleotide. Polymerases mayuse RNA templates, e.g., be RNA-dependent, or DNA templates, e.g., be DNA-dependent. Additionally, polymerases may utilize ribonucleotides to synthesize RNA polynucleotides or utilize deoxyribonucleotides to synthesize DNA polynucleotides. In some embodiments, a polymerase is a reverse transcriptase.Polymerase Enzymes

[0039] In some embodiments, the present disclosure relates to methods of enhancing the reverse transcription and template switching activity of a polymerase by separating the reactions into individual steps. Polymerases are enzymes that catalyze phosphodi ester bond formation between nucleotides, e.g., ribonucleotides or deoxyribonucleotides, to generate a polynucleotide product, e.g., a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA), in a sequence- directed manner. For example, polymerases synthesize polynucleotide products that are complementary in sequence to a template nucleic acid, e.g., a template RNA molecule or template DNA molecule. Polymerases are categorized by the type of nucleic acid synthesized and the type of nucleic acid used as a template, e.g., RNA or DNA. For example, polymerases can be RNA polymerases, e.g., synthesize RNA products, or DNA polymerases, e.g., synthesize DNA products. As a further example, polymerases can be RNA-dependent, e.g., use RNA as a template, or DNA-dependent, e.g., use DNA as a template. Thus, polymerases are generally categorized as being RNA-dependent RNA polymerases, RNA-dependent DNA polymerases, DNA-dependent RNA polymerases, or DNA-dependent DNA polymerases. In some embodiments, the present disclosure relates to RNA-dependent DNA polymerases, e.g., reverse transcriptases.

[0040] Reverse transcriptases are enzymes that synthesize single-stranded complementary DNA (cDNA) from a single-stranded RNA template. Reverse transcriptases are useful for methods of analyzing gene expression by measuring abundance of messenger RNA (mRNA) such as reverse transcription coupled to quantitative polymerase chain reaction (qRT- PCR) or RNA sequencing (RNA-seq). However, the accuracy of these methods can often be limited by the ability of a reverse transcriptase to reverse transcribe long template RNAs or RNAs with complex secondary structures from end-to-end, e.g., the ability of the reverse transcriptase to synthesize full-length cDNAs, e.g., the processivity of the reverse transcriptase. Additionally, methods for measuring gene expression can further be limited by the ability of areverse transcriptase to reverse transcribe low-abundance RNAs. For example, certain RTs are unable to amplify meaningful amounts of low-abundance RNAs in complex mixtures, which can impact their utility in a research or commercial setting, e.g., in methods of measuring gene expression from samples with low amounts of input RNA, such as single-cell RNA sequencing or in situ RNA sequencing. The present disclosure provides methods for improving the ability of reverse transcriptases to synthesize cDNAs, e.g., full-length cDNAs, from long or complex RNA templates, e.g., improving the processivity of the reverse transcriptase, or enhancing the ability of reverse transcriptase to synthesize cDNA from RNAs with low abundance in a sample, by including an anionic polymer into the RT reaction mixture.

[0041] The methods described herein for enhancing reverse transcription and template switching activity apply to any polymerase, e.g., any polymerase having reverse transcription activity, e.g., a reverse transcriptase, known in the art. For example, the polymerase may be a DNA polymerase or a reverse transcriptase. In some embodiments, the polymerase is a DNA polymerase, e.g., a DNA polymerase having reverse transcription activity. In some embodiments, the DNA polymerase is Thermits aquaticus (Taq) polymerase. In some embodiments, the DNA polymerase is Thermus thermophilus (Tth) polymerase. In some embodiments, the polymerase is human DNA polymerase r| (Pol eta). In some embodiments, the DNA polymerase is a DNA polymerase engineered to have reverse transcription activity. In some embodiments, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to a sequence listed in Table 1, e.g., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to a sequence listed in Table 1. In some embodiments, the reverse transcriptase consists of a sequence listed in Table 1.

[0042] In some embodiments, the polymerase is a reverse transcriptase. In some embodiments, the reverse transcriptase is derived from a virus, an intron, a telomerase, or a retrotransposon. In some embodiments, the reverse transcriptase is derived from a telomerase, e.g., a mammalian telomerase. In some embodiments, the reverse transcriptase is derived from a mobile genetic element, e g., a retrotransposon, e.g., a plant or animal retrotransposon, e.g., a non-long terminal repeat (non-LTR) retrotransposon. In some embodiments, the reverse transcriptase is derived from a mobile genetic element, e.g., a self-splicing intron, e.g., a group II intron. In some embodiments, the reverse transcriptase has a sequence listed in Table 1. In some embodiments, the reverse transcriptase has a sequence with at least 60% to 99.9% identity to asequence listed in Table 1 , e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 99.5%, or 99.9% or greater identity to a sequence listed in Table 1.Table 1. Exemplary reverse transcriptase sequences.

[0043] In some embodiments, the reverse transcriptase comprises a sequence with about % to about 99.9% identity to any one of SEQ ID NOs: 1-15, e.g., at least about 60%, 65%, %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity toany one of SEQ ID NOs: 1-15. In some embodiments, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to any one of SEQ ID NOs: 1 or 2, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to any one of SEQ ID NOs: 1 or 2. In some embodiments, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to any one of SEQ ID NOs: 14 or 15, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to any one of SEQ ID NOs: 14 or 15. In some embodiments, the reverse transcriptase comprises any one of SEQ ID NOs: 1-15. In some embodiments, the reverse transcriptase comprises any one of SEQ ID NOs: 1 or 2. In some embodiments, the reverse transcriptase comprises any one of SEQ ID NOs: 14 or 15. In some embodiments, the reverse transcriptase consists of any one of SEQ ID NOs: 1-15. In some embodiments, the reverse transcriptase consists of any one of SEQ ID NOs: 1 or 2. In some embodiments, the reverse transcriptase consists of any one of SEQ ID NOs: 14 or 15.

[0044] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 1, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 1. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO:1. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO: 1. In an embodiment, the reverse transcriptase comprises a sequence with about 90% identity to SEQ ID NO: 1. In an embodiment, the reverse transcriptase comprises a sequence with about 95% identity to SEQ ID NO: 1. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO: 1. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO: 1. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO: 1. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO:1. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO: 1. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO: 1. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 1. In an embodiment, MarathonRT™ comprises the amino acid sequence of SEQ ID NO: 1, e.g., or a fragment or variant thereof. In an embodiment, MarathonRT™ comprises theamino acid sequence of SEQ ID NO: 1 . In an embodiment, the amino acid sequence of SEQ ID NO: 1 comprises the full-length sequence of MarathonRT™.

[0045] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 2, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 2. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO:2. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO:2. In an embodiment, the reverse transcriptase comprises a sequence with about 90% identity to SEQ ID NO:2. In an embodiment, the reverse transcriptase comprises a sequence with about 95% identity to SEQ ID NO:2. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO:2. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO:2. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO:2. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO:2. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO:2. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO:2. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 2. In an embodiment, MarathonRT™ comprises the amino acid sequence of SEQ ID NO: 2, e.g., or a fragment or variant thereof. In an embodiment, MarathonRT™ comprises the amino acid sequence of SEQ ID NO: 2. In an embodiment, the amino acid sequence of SEQ ID NO: 2 comprises a domain of MarathonRT™, for example, the reverse transcriptase (RT) domain. In an embodiment, the amino acid sequence of SEQ ID NO: 2 comprises the finger subdomain and the palm subdomain of MarathonRT™. In an embodiment, the amino acid sequence of SEQ ID NO: 2 comprises the reverse transcriptase domain of MarathonRT™.

[0046] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 3, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 3. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO:3. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO:3. In an embodiment, the reverse transcriptase comprises a sequence with about 90% identity to SEQ ID NO:3. In an embodiment, the reverse transcriptase comprises a sequencewith about 95% identity to SEQ ID NO:3. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO:3. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO:3. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO:3. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO:3. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO:3. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO:3. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 3. In an embodiment, Roseburia intestinalis reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 3, e.g., or a fragment or variant thereof. In an embodiment, Roseburia intestinalis reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 3. In an embodiment, the amino acid sequence of SEQ ID NO: 3 comprises the full-length sequence of Roseburia intestinalis reverse transcriptase.

[0047] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 4, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 4. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO:4. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO:4. In an embodiment, the reverse transcriptase comprises a sequence with about 90% identity to SEQ ID NO:4. In an embodiment, the reverse transcriptase comprises a sequence with about 95% identity to SEQ ID NO:4. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO:4. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO:4. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO:4. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO:4. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO:4. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO:4. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 4. In an embodiment, Roseburia intestinalis reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 4, e.g., or a fragment or variant thereof. In an embodiment, Roseburia intestinalis reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 4.In an embodiment, the amino acid sequence of SEQ ID NO: 4 comprises a domain of Roseburia intestinalis reverse transcriptase, for example, the reverse transcriptase (RT) domain. In an embodiment, the amino acid sequence of SEQ ID NO: 4 comprises the reverse transcriptase domain of Roseburia intestinalis reverse transcriptase.

[0048] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 5, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 5. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO:5. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO:5. In an embodiment, the reverse transcriptase comprises a sequence with about 90% identity to SEQ ID NO:5. In an embodiment, the reverse transcriptase comprises a sequence with about 95% identity to SEQ ID NO:5. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO:5. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO:5. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO: 5. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO:5. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO:5. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO:5. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 5. In an embodiment, Moloney murine leukemia virus (MMLV) reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 5, e.g., or a fragment or variant thereof. In an embodiment, MMLV reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 5. In an embodiment, the amino acid sequence of SEQ ID NO: 5 comprises the full- length sequence of MMLV reverse transcriptase.

[0049] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 6, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 6. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO:6. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO:6. In an embodiment, the reverse transcriptase comprises a sequence with about 90% identity to SEQ ID NO:6. In an embodiment, the reverse transcriptase comprises a sequencewith about 95% identity to SEQ ID NO:6. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO:6. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO:6. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO:6. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO:6. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO:6. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO:6. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 6. In an embodiment, MMLV reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 6, e.g., or a fragment or variant thereof. In an embodiment, MMLV reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 6. In an embodiment, the amino acid sequence of SEQ ID NO: 6 comprises a domain of MMLV reverse transcriptase, for example, the reverse transcriptase (RT) domain. In an embodiment, the amino acid sequence of SEQ ID NO: 6 comprises the reverse transcriptase domain of MarathonRT™.

[0050] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 7, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 7. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO:7. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO: 7. In an embodiment, the reverse transcriptase comprises a sequence with about 90% identity to SEQ ID NO:7. In an embodiment, the reverse transcriptase comprises a sequence with about 95% identity to SEQ ID NO:7. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO:7. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO:7. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO:7. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO:7. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO:7. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO:7. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 7. In an embodiment, Bombyx mori R2 element reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 7, e g., or a fragment or variant thereof. In an embodiment,Bombyx mori R2 element reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 7. In an embodiment, the amino acid sequence of SEQ ID NO: 7 comprises the full-length sequence of Bombyx mori R2 element reverse transcriptase.

[0051] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 8, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 8. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO:8. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO:8. In an embodiment, the reverse transcriptase comprises a sequence with about 90% identity to SEQ ID NO:8. In an embodiment, the reverse transcriptase comprises a sequence with about 95% identity to SEQ ID NO:8. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO:8. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO: 8. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO: 8. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO:8. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO:8. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO:8. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 8. In an embodiment, Bombyx mori R2 element reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 8, e g., or a fragment or variant thereof. In an embodiment, Bombyx mori R2 element reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 8. In an embodiment, the amino acid sequence of SEQ ID NO: 8 comprises a domain of Bombyx mori R2 element reverse transcriptase, for example, the reverse transcriptase (RT) domain. In an embodiment, the amino acid sequence of SEQ ID NO: 8 comprises the reverse transcriptase domain of Bombyx mori R2 element reverse transcriptase.

[0052] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 9, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 9. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO:9. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO: 9. In an embodiment, the reverse transcriptase comprises a sequence with about90% identity to SEQ ID NON. In an embodiment, the reverse transcriptase comprises a sequence with about 95% identity to SEQ ID NO:9. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO:9. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO:9. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO:9. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO:9. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO:9. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO:9. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 9. In an embodiment, avian myeloblastosis virus (AMV) reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 9, e.g., or a fragment or variant thereof. In an embodiment, AMV reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 9. In an embodiment, the amino acid sequence of SEQ ID NO: 9 comprises the full-length sequence of AMV reverse transcriptase.

[0053] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 10, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 10. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO: 10. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO: 10. In an embodiment, the reverse transcriptase comprises a sequence with about 90% identity to SEQ ID NO: 10. In an embodiment, the reverse transcriptase comprises a sequence with about 95% identity to SEQ ID NO: 10. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO: 10. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO: 10. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO: 10. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO: 10. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO: 10. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO: 10. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 10. In an embodiment, AMV reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 10, e.g., or a fragmentor variant thereof. In an embodiment, AMV reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 10. In an embodiment, the amino acid sequence of SEQ ID NO: 10 comprises a domain of AMV reverse transcriptase, for example, the reverse transcriptase (RT) domain. In an embodiment, the amino acid sequence of SEQ ID NO: 10 comprises the reverse transcriptase domain of AMV reverse transcriptase.

[0054] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 11, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 11. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO:11. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO: 11. In an embodiment, the reverse transcriptase comprises a sequence with about 90% identity to SEQ ID NO: 11. In an embodiment, the reverse transcriptase comprises a sequence with about 95% identity to SEQ ID NO:11. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO: 11. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO:11. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO: 11. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO: 11. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO: 11. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO: 11. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 11. In an embodiment, human telomerase (TERT) reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 11, e.g., or a fragment or variant thereof. In an embodiment, TERT reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 11. In an embodiment, the amino acid sequence of SEQ ID NO: 11 comprises the full-length sequence of TERT reverse transcriptase.

[0055] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 12, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 12. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO: 12. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO: 12. In an embodiment, the reverse transcriptase comprises a sequencewith about 90% identity to SEQ ID NO: 12. In an embodiment, the reverse transcriptase comprises a sequence with about 95% identity to SEQ ID NO: 12. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO: 12. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO: 12. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO: 12. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO: 12. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO: 12. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO: 12. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 12. In an embodiment, TERT reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 12, e.g., or a fragment or variant thereof. In an embodiment, TERT reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 12. In an embodiment, the amino acid sequence of SEQ ID NO: 12 comprises a domain of TERT reverse transcriptase, for example, the reverse transcriptase (RT) domain. In an embodiment, the amino acid sequence of SEQ ID NO: 12 comprises the reverse transcriptase domain of TERT reverse transcriptase.

[0056] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 13, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 13. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO:13. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO: 13. In an embodiment, the reverse transcriptase comprises a sequence with about 90% identity to SEQ ID NO: 13. In an embodiment, the reverse transcriptase comprises a sequence with about 95% identity to SEQ ID NO:13. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO: 13. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO: 13. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO: 13. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO: 13. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO: 13. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO: 13. In anembodiment, the reverse transcriptase consists of SEQ ID NO: 13. In an embodiment, TGIRT™ reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 13, e.g., or a fragment or variant thereof. In an embodiment, TGIRT™ reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 13. In an embodiment, the amino acid sequence of SEQ ID NO: 13 comprises the full-length sequence of TGIRT™ reverse transcriptase.

[0057] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 14, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 14. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO: 14. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO: 14. In an embodiment, the reverse transcriptase comprises a sequence with about 90% identity to SEQ ID NO: 14. In an embodiment, the reverse transcriptase comprises a sequence with about 95% identity to SEQ ID NO: 14. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO: 14. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO: 14. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO: 14. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO: 14. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO: 14. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO: 14. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 14. In an embodiment, UltraMarathonRT® comprises the amino acid sequence of SEQ ID NO: 14, e.g., or a fragment or variant thereof. In an embodiment, UltraMarathonRT® comprises the amino acid sequence of SEQ ID NO: 14. In an embodiment, the amino acid sequence of SEQ ID NO: 14 comprises the full-length sequence of UltraMarathonRT®.

[0058] In an embodiment, the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to SEQ ID NO: 15, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to SEQ ID NO: 15. In an embodiment, the reverse transcriptase comprises a sequence with about 80% identity to SEQ ID NO: 15. In an embodiment, the reverse transcriptase comprises a sequence with about 85% identity to SEQ ID NO: 15. In an embodiment, the reverse transcriptase comprises a sequencewith about 90% identity to SEQ ID NO: 15. In an embodiment, the reverse transcriptase comprises a sequence with about 95% identity to SEQ ID NO:15. In an embodiment, the reverse transcriptase comprises a sequence with about 96% identity to SEQ ID NO: 15. In an embodiment, the reverse transcriptase comprises a sequence with about 97% identity to SEQ ID NO: 15. In an embodiment, the reverse transcriptase comprises a sequence with about 98% identity to SEQ ID NO: 15. In an embodiment, the reverse transcriptase comprises a sequence with about 99% identity to SEQ ID NO: 15. In an embodiment, the reverse transcriptase comprises a sequence with about 99.5% identity to SEQ ID NO: 15. In an embodiment, the reverse transcriptase comprises a sequence with about 99.9% identity to SEQ ID NO: 15. In an embodiment, the reverse transcriptase consists of SEQ ID NO: 15. In an embodiment, UltraMarathonRT® comprises the amino acid sequence of SEQ ID NO: 15, e.g., or a fragment or variant thereof. In an embodiment, UltraMarathonRT® comprises the amino acid sequence of SEQ ID NO: 15. In an embodiment, the amino acid sequence of SEQ ID NO: 15 comprises a domain of UltraMarathonRT®, for example, the reverse transcriptase (RT) domain. In an embodiment, the amino acid sequence of SEQ ID NO: 15 comprises the finger subdomain and the palm subdomain of UltraMarathonRT®. In an embodiment, the amino acid sequence of SEQ ID NO: 15 comprises the reverse transcriptase domain of UltraMarathonRT®. In some embodiments, the reverse transcriptase is derived from a mobile genetic element. For example, the reverse transcriptase may be derived from a non-long terminal repeat (non-LTR) retrotransposon or a group II intron. In some embodiments, the reverse transcriptase is a non- LTR retrotransposon reverse transcriptase. In some embodiments, the non-LTR retrotransposon reverse transcriptase is a Bombyx mori R2 RNA element reverse transcriptase. In some embodiments, the non-LTR retrotransposon reverse transcriptase is a human LI element reverse transcriptase. In some embodiments, the reverse transcriptase is a group II intron reverse transcriptase, e.g., a maturase reverse transcriptase. In some embodiments, the group II intron reverse transcriptase is a maturase. In some embodiments, the group II intron reverse transcriptase is a maturase encoded by Eubacterium rectale. In some embodiments, the group II intron reverse transcriptase is a maturase encoded by Roseburia intestinalis . In some embodiments, the group II intron reverse transcriptase is MarathonRT™ reverse transcriptase. In some embodiments, the group II intron reverse transcriptase is UltraMarathonRT®. In someembodiments, the group TI intron reverse transcriptase is Induro® reverse transcriptase. In some embodiments, the group II intron reverse transcriptase is a TGIRT™ reverse transcriptase.

[0059] In some embodiments, the reverse transcriptase is derived from a virus. For example, the reverse transcriptase may be derived from a retrovirus. In some embodiments, the reverse transcriptase is an Avian Myeloblastosis Virus (AMV) reverse transcriptase. In some embodiments, the reverse transcriptase is a Human Immunodeficiency Virus reverse transcriptase. In some embodiments, the reverse transcriptase is a Rous Sarcoma Virus reverse transcriptase. In some embodiments, the reverse transcriptase is a Moloney Murine Leukemia Virus (MMLV) reverse transcriptase. In some embodiments, the MMLV reverse transcriptase is Maxima™ H Minus reverse transcriptase. In some embodiments, the MMLV reverse transcriptase is SuperScript™ II reverse transcriptase. In some embodiments, the MMLV reverse transcriptase is SuperScript™ III reverse transcriptase. In some embodiments, the MMLV reverse transcriptase is SuperScript™ IV reverse transcriptase. In some embodiments, the MMLV reverse transcriptase is PrimeScript™ reverse transcriptase. In some embodiments, the MMLV reverse transcriptase is GoScript™ reverse transcriptase. In some embodiments, the MMLV reverse transcriptase is ProtoScript® II reverse transcriptase. In some embodiments, the MMLV reverse transcriptase is SMARTScribe™ reverse transcriptase.

[0060] In another aspect, the present disclosure features ultraprocessive reverse transcriptases. In some embodiments, the reverse transcriptase is an ultraprocessive reverse transcriptase, e.g., a reverse transcriptase capable of synthesizing cDNAs of 4,000 nucleotides, 5,000 nucleotides, 6,000 nucleotides, 7,000 nucleotides, 8,000 nucleotides, 9,000 nucleotides, 10,000 nucleotides, 11,000 nucleotides, 12,000 nucleotides, 15,000 nucleotides, 20,000 nucleotides, 25,000 nucleotides, or 30,000 nucleotides or more in length. In some embodiments, the ultraprocessive reverse transcriptase is MarathonRT™. In some embodiments, the ultraprocessive reverse transcriptase is UltraMarathonRT®.Oligonucleotide Primers

[0061] The methods described herein for enhancing reverse transcription and template switching may further include using an oligonucleotide primer, e.g., a plurality of oligonucleotide primers, that contain many sequence elements. In some embodiments, the reverse transcription reaction mixture comprises at least two oligonucleotide primers, e.g., atleast 2, 3, or 4 oligonucleotide primers. In some embodiments, the oligonucleotide primer is between about 5 and about 250 nucleotides in length, e.g., about 5 and about 200, about 5 and about 150, about 5 and about 100, about 5 and about 75, about 5 and about 50, and about 5 and about 25 nucleotides in length. In some embodiments, the oligonucleotide primer is between about 5 and about 100 nucleotides in length. In some embodiments, the oligonucleotide primer is about 5 nucleotides in length. In some embodiments, the oligonucleotide primer is about 10 nucleotides in length. In some embodiments, the oligonucleotide primer is about 20 nucleotides in length. In some embodiments, the oligonucleotide primer is about 25 nucleotides in length. In some embodiments, the oligonucleotide primer is about 35 nucleotides in length. In some embodiments, the oligonucleotide primer is about 40 nucleotides in length. In some embodiments, the oligonucleotide primer is about 45 nucleotides in length. In some embodiments, the oligonucleotide primer is about 50 nucleotides in length. In some embodiments, the oligonucleotide primer is about 55 nucleotides in length. In some embodiments, the oligonucleotide primer is about 60 nucleotides in length. In some embodiments, the oligonucleotide primer is about 65 nucleotides in length. In some embodiments, the oligonucleotide primer is about 70 nucleotides in length. In some embodiments, the oligonucleotide primer is about 75 nucleotides in length. In some embodiments, the oligonucleotide primer is about 80 nucleotides in length. In some embodiments, the oligonucleotide primer is about 85 nucleotides in length. In some embodiments, the oligonucleotide primer is about 90 nucleotides in length. In some embodiments, the oligonucleotide primer is about 95 nucleotides in length. In some embodiments, the oligonucleotide primer is about 100 nucleotides in length. In some embodiments, the oligonucleotide primer is about 125 nucleotides in length. In some embodiments, the oligonucleotide primer is about 150 nucleotides in length. In some embodiments, the oligonucleotide primer is about 200 nucleotides in length. In some embodiments, the oligonucleotide primer is about 250 nucleotides in length. In some embodiments, the oligonucleotide primer is about 500 nucleotides in length. In some embodiments, the oligonucleotide primer is about 750 nucleotides in length.

[0062] The oligonucleotide primer may be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). In some embodiments, the oligonucleotide primer is DNA. In some embodiments, the oligonucleotide primer is RNA. In some embodiments the oligonucleotideprimer comprises a naturally occurring nucleotide or a non-naturally occurring nucleotide. In some embodiments, the oligonucleotide primer comprises one or more naturally occurring nucleotides. In some embodiments, the oligonucleotide primer comprises one or more non- naturally occurring nucleotides.

[0063] In some embodiments, the oligonucleotide primer comprises an adenine (A), thymine (T), guanine (G), cytosine (C), or uracil (U) nucleotide. In some embodiments, the oligonucleotide primer comprises between 1 and about 30 adenine nucleotides, e.g., about 1 and about 25, about 1 and about 20, about 1 and about 15, about 1 and about 10, and about 1 and about 5 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 1 adenine nucleotide. In some embodiments, the oligonucleotide primer comprises 2 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 3 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 4 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 5 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 6 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 7 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 8 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 9 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 10 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 11 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 12 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 13 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 14 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 15 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 16 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 17 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 18 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 19 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 20 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 21 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 22 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 23 adenine nucleotides. In someembodiments, the oligonucleotide primer comprises 24 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 25 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 26 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 27 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 28 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 29 adenine nucleotides. In some embodiments, the oligonucleotide primer comprises 30 adenine nucleotides.

[0064] In some embodiments, the oligonucleotide primer comprises between 1 and about 30 thymine nucleotides, e.g., about 1 and about 25, about 1 and about 20, about 1 and about 15, about 1 and about 10, and about 1 and about 5 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 1 thymine nucleotide. In some embodiments, the oligonucleotide primer comprises 2 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 3 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 4 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 5 thymine nucleotides. In some embodiments, the oligonucleotide primer com comprises 6 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 7 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 8 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 9 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 10 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 11 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 12 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 13 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 14 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 15 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 16 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 17 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 18 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 19 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 20 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 21 thymine nucleotides. In some embodiments, theoligonucleotide primer comprises 22 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 23 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 24 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 25 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 26 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 27 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 28 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 29 thymine nucleotides. In some embodiments, the oligonucleotide primer comprises 30 thymine nucleotides.

[0065] In some embodiments, the oligonucleotide primer comprises between 1 and about 30 cytosine nucleotides, e.g., about 1 and about 25, about 1 and about 20, about 1 and about 15, about 1 and about 10, and about 1 and about 5 cytosine nucleotides. In some embodiments, the oligonucleotide primer has 1 cytosine nucleotide. In some embodiments, the oligonucleotide primer comprises 2 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 3 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 4 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 5 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 6 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 7 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 8 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 9 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 10 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 11 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 12 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 13 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 14 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 15 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 16 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 17 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 18 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 19 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 20 cytosine nucleotides. In someembodiments, the oligonucleotide primer comprises 21 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 22 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 23 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 24 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 25 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 26 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 27 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 28 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 29 cytosine nucleotides. In some embodiments, the oligonucleotide primer comprises 30 cytosine nucleotides.

[0066] In some embodiments, the oligonucleotide primer comprises between 1 and about 30 uracil nucleotides, e g., about 1 and about 25, about 1 and about 20, about 1 and about 15, about 1 and about 10, and about 1 and about 5 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 1 uracil nucleotide. In some embodiments, the oligonucleotide primer comprises 2 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 3 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 4 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 5 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 6 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 7 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 8 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 9 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 10 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 11 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 12 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 13 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 14 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 15 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 16 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 17 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 18 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 19 uracil nucleotides. In some embodiments, theoligonucleotide primer comprises 20 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 21 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 22 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 23 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 24 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 25 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 26 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 27 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 28 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 29 uracil nucleotides. In some embodiments, the oligonucleotide primer comprises 30 uracil nucleotides.

[0067] In some embodiments, the oligonucleotide primer comprises between 1 and about 30 guanosine nucleotides, e.g., about 1 and about 25, about 1 and about 20, about 1 and about 15, about 1 and about 10, and about 1 and about 5 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 1 guanosine nucleotide. In some embodiments, the oligonucleotide primer comprises 2 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 3 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 4 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 5 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 6 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 7 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 8 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 9 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 10 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 11 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 12 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 13 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 14 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 15 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 16 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 17 guanosine nucleotides. In some embodiments, theoligonucleotide primer comprises 18 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 19 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 20 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 21 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 22 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 23 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 24 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 25 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 26 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 27 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 28 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 29 guanosine nucleotides. In some embodiments, the oligonucleotide primer comprises 30 guanosine nucleotides.

[0068] In an embodiment, the reaction mixture comprises a plurality of oligonucleotide primers, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more oligonucleotide primers. In some embodiments, the reaction mixture comprises 2 oligonucleotide primers. In some embodiments, the reaction mixture comprises 3 oligonucleotide primers. In some embodiments, the oligonucleotide primer is a template-switching oligonucleotide primer.

[0069] In some embodiments, the oligonucleotide primer comprises a modification, e g., a chemical modification to the nucleobase, ribose, 5’ terminus, or 3’ terminus. For example, the oligonucleotide primer may comprise a modification that prevents non-specific primer extension, annealing between oligonucleotide primers, or concatemerization. In some embodiments, the modification is a DNA / RNA hybrid wherein a DNA sequence is covalently linked to an RNA sequence. In some embodiments, the modification is a non-natural nucleotide or a nucleotide analog. In some embodiments, the oligonucleotide primer comprises a chemically modified nucleobase. In some embodiments, the oligonucleotide primer comprises a chemically modified ribose. In some embodiments, the oligonucleotide primer comprises a chemically modified 5’ terminus. In some embodiments, the oligonucleotide primer comprises a chemically modified 3’ terminus.

[0070] In some embodiments, the oligonucleotide primer is a reverse transcription primer, e.g., a primer used for a reverse transcription reaction. For example, a reversetranscription primer may be used by a polymerase, e.g., a reverse transcriptase, to synthesize cDNA. In some embodiments, the reverse transcription primer is complementary to the 3’ end of the target RNA, e.g., complementary to 3’ most 10, 20, 50, 100, 150, 200 or more nucleotides of the target RNA. In some embodiments, the reverse transcription primer is at least partially complementary to the polyAtail of a target RNA, e.g., the polyAtail of a messenger RNA (mRNA).

[0071] In some embodiments, the oligonucleotide primer is a template switching oligonucleotide (TSO). The TSO may comprise a 3’ end that is complementary to the product of non-templated nucleotide addition by a reverse transcriptase to cDNA synthesized by the reverse transcriptase. For example, the 3’ terminal nucleotides of the TSO may base pair with the 3’ terminal nucleotides of the cDNA strand, wherein the 3’ terminal nucleotides of the cDNA strand are synthesized by non-templated nucleotide addition activity of a reverse transcriptase. In some embodiments, the 3’ end of the TSO comprises three nucleotides that are complementary to three nucleotides added to the cDNA strand by non-templated nucleotide addition. In some embodiments, the 3’ end of the TSO comprises two nucleotides that are complementary to two nucleotides added to the cDNA strand by non-templated nucleotide addition. In some embodiments, the 3’ end of the TSO comprises one nucleotide that is complementary to one nucleotide added to the cDNA strand by non-templated nucleotide addition. In some embodiments, the 3’ end of the TSO comprises three terminal T nucleotides. In some embodiments, the 3’ end of the TSO comprises three terminal U nucleotides. In some embodiments, the 3’ end of the TSO comprises three G nucleotides. In some embodiments, the 3’ end of the TSO comprises one T nucleotide. In some embodiments, the 3’ end of the TSO comprises one U nucleotide. In some embodiments, the 3’ end of the TSO comprises one A nucleotide. In some embodiments, the 3’ end of the TSO comprises one G nucleotide. In some embodiments, the 3’ end of the TSO comprises one C nucleotide.Input and Target RNA

[0072] In one aspect, the present disclosure features a reverse transcription reaction mixture comprising input RNA, e.g., substrate RNA for a polymerase, e.g., template RNA for cDNA synthesis. For example, the input RNA may comprise target RNA, e.g., RNA for which a value is acquired using a method of reverse transcription. In some embodiments, the input RNAcomprises a mixture of a plurality of RNAs, e.g., a mixture of RNAs having identical or nonidentical sequences. In some embodiments, the input RNA comprises a mixture of RNAs consisting of identical sequences. In some embodiments, the input RNA comprises a mixture of RNAs consisting of non-identical sequences. In some embodiments, the source of the input RNA is a cell or a source other than a cell. In some embodiments, the source of the input RNA is a cell, e.g., a eukaryotic cell or a prokaryotic cell. In some embodiments, the eukaryotic cell is an animal cell, fungal cell, or a plant cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the prokaryotic cell is a bacterial cell or an archaeal cell. In some embodiments, the source of the input RNA is a virus. In some embodiments, the source of the input RNA is a single cell or a plurality of cells. In some embodiments, the source of the input RNA is a single cell. In some embodiments, the source of the input RNA is a plurality of cells. In some embodiments, the input RNA comprises total cellular RNA, e.g., ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA (miRNA), or any RNA present in a cell. In some embodiments, the input RNA is rRNA. In some embodiments, the input RNA is tRNA. In some embodiments, the input RNA is mRNA. In some embodiments, the input RNA is ncRNA. In some embodiments, the input RNA is miRNA. In some embodiments, the input RNA is about 5 to about 2,500,000 nucleotides in length, e.g., about 5 nucleotides, 50 nucleotides, 100 nucleotides, 500 nucleotides, 1,000 nucleotides, 5,000 nucleotides, 7,000 nucleotides, 10,000 nucleotides, 15,000 nucleotides, 20,000 nucleotides, 50,000 nucleotides, 100,000 nucleotides, 500,000 nucleotides, 1,000,000 nucleotides, or 2,500,000 nucleotides, or more in length. In some embodiments, the input RNA is about 5 nucleotides in length. In some embodiments, the input RNA is about 50 nucleotides in length. In some embodiments, the input RNA is about 100 nucleotides in length. In some embodiments, the input RNA is about 500 nucleotides in length. In some embodiments, the input RNA is about 1,000 nucleotides in length. In some embodiments, the input RNA is about 5,000 nucleotides in length. In some embodiments, the input RNA is about 7,000 nucleotides in length. In some embodiments, the input RNA is about 10,000 nucleotides in length. In some embodiments, the input RNA is about 15,000 nucleotides in length. In some embodiments, the input RNA is about 20,000 nucleotides in length. In some embodiments, the input RNA is about 50,000 nucleotides in length. In some embodiments, the input RNA is about 100,000 nucleotides in length. In some embodiments, the input RNA is about 500,000 nucleotides in length. In some embodiments, theinput RNA is about 1 ,000,000 nucleotides in length. In some embodiments, the input RNA is about 2,500,000 nucleotides in length. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is from about 1 pg to about 10 fg, e.g., the level of input RNA is about 1 pg, 500 ng, 250 ng, 100 ng, 50 ng, 25 ng, 10 ng, 5 ng, 1 ng, 500 pg, 250 pg, 100 pg, 50 pg, 25 pg, 10 pg, 5 pg, 1 pg, 500 fg, 250 fg, 100 fg, 50 fg, 25 fg, 10 fg, or less. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 1 pg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 500 ng. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 250 ng. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 100 ng. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 50 ng. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 25 ng. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 10 ng. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 5 ng. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 1 ng. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 500 pg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 250 pg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 100 pg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 50 pg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 25 pg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 10 pg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 5 pg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 1 pg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 500 fg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 250 fg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 100 fg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 50 fg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 25 fg. In some embodiments, the level of input RNA in the reverse transcription reaction mixture is about 10 fg.In some embodiments, the concentration of input RNA in the reverse transcription reaction mixture is from about 0.001 zeptomolar (zM) to about 1 micromolar (pM), e.g., 0.001 zM, 0.01 zM, 0.1 zM, 1 zM, 0.01 attomolar (aM), 0.1 aM, 1 aM, 0.01 femtomolar (fM), 0.1 fM, 1 fM, 0.01 picomolar (pM), 0.1 pM, 1 pM, 0.01 nanomolar (nM), 0.1 nM, 1 nM, 0.01 pM, 0.1 pM, or 1 pM or greater. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 0.001 zM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 0.01 zM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 0.1 zM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 1 zM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 0.01 aM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 0.1 aM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 1 aM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 0.01 fM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 0.1 fM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 1 fM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 0.01 pM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 0.1 pM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 1 pM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 0.01 nM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 0.1 nM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 1 nM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 0.01 pM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 0.1 pM. In some embodiments, the concentration of input RNA in a reverse transcription reaction mixture is about 1 pM.

[0073] In some embodiments, the input RNA comprises target RNA, e.g., an RNA sequence detected in the reverse transcription reaction. In some embodiments, the source of the target RNA is a cell or a source other than a cell. In some embodiments, the source of the target RNA is a cell, e.g., a eukaryotic cell or a prokaryotic cell. In some embodiments, the eukaryoticcell is an animal cell, fungal cell, or a plant cell. Tn some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the prokaryotic cell is a bacterial cell or an archaeal cell. In some embodiments, the source of the target RNAis a virus. In some embodiments, the source of the target RNA is a single cell or a plurality of cells. In some embodiments, the source of the target RNA is a single cell. In some embodiments, the source of the target RNA is a plurality of cells. In some embodiments, the target RNA comprises total cellular RNA, e.g., ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA (miRNA), or any RNA present in a cell. In some embodiments, the target RNA is rRNA. In some embodiments, the target RNA is tRNA. In some embodiments, the target RNA is mRNA. In some embodiments, the target RNA is ncRNA. In some embodiments, the target RNA is miRNA. In some embodiments, the target RNA is about 5 to about 2,500,000 nucleotides in length, e g., about 5 nucleotides, 50 nucleotides, 100 nucleotides, 500 nucleotides, 1,000 nucleotides, 5,000 nucleotides, 7,000 nucleotides, 10,000 nucleotides, 15,000 nucleotides, 20,000 nucleotides, 50,000 nucleotides, 100,000 nucleotides, 500,000 nucleotides, 1,000,000 nucleotides, or 2,500,000 nucleotides, or more in length. In some embodiments, the target RNA is about 5 nucleotides in length. In some embodiments, the target RNA is about 50 nucleotides in length. In some embodiments, the target RNA is about 100 nucleotides in length. In some embodiments, the target RNA is about 500 nucleotides in length. In some embodiments, the target RNA is about 1,000 nucleotides in length. In some embodiments, the target RNA is about 5,000 nucleotides in length. In some embodiments, the target RNAis about 7,000 nucleotides in length. In some embodiments, the target RNA is about 10,000 nucleotides in length. In some embodiments, the target RNA is about 15,000 nucleotides in length. In some embodiments, the target RNA is about 20,000 nucleotides in length. In some embodiments, the target RNA is about 50,000 nucleotides in length. In some embodiments, the target RNA is about 100,000 nucleotides in length. In some embodiments, the target RNA is about 500,000 nucleotides in length. In some embodiments, the target RNA is about 1,000,000 nucleotides in length. In some embodiments, the target RNA is about 2,500,000 nucleotides in length. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is from about 1 pg to about 10 fg, e.g., the level of input RNA is about 1 pg, 500 ng, 250 ng, 100 ng, 50 ng, 25 ng, 10 ng, 5 ng, 1 ng, 500 pg, 250 pg, 100 pg, 50 pg, 25 pg, 10 pg, 5 pg, 1 pg, 500 fg, 250 fg, 100 fg, 50 fg, 25 fg, 10 fg, or less. In some embodiments, the level of target RNA in the reverse transcription reactionmixture is about 1 pig. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 500 ng. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 250 ng. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 100 ng. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 50 ng. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 25 ng. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 10 ng. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 5 ng. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 1 ng. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 500 pg. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 250 pg. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 100 pg. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 50 pg. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 25 pg. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 10 pg. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 5 pg. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 1 pg. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 500 fg. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 250 fg. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 100 fg. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 50 fg. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 25 fg. In some embodiments, the level of target RNA in the reverse transcription reaction mixture is about 10 fg. In some embodiments, the concentration of target RNA in the reverse transcription reaction mixture is from about 0.001 zeptomolar (zM) to about 1 micromolar (pM), e.g., 0.001 zM, 0.01 zM, 0.1 zM, 1 zM, 0.01 attomolar (aM), 0.1 aM, 1 aM, 0.01 femtomolar (IM), 0.1 IM, 1 0.01 picomolar (pM), 0.1 pM, 1 pM, 0.01 nanomolar (nM), 0.1 nM, 1 nM, 0.01 pM, 0.1 pM, or 1 pM or greater. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 0.001 zM. In some embodiments, the concentration of target RNA in areverse transcription reaction mixture is about 0.01 zM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 0.1 zM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 1 zM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 0.01 aM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 0.1 aM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 1 aM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 0.01 fM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 0.1 fM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 1 fM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 0.01 pM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 0.1 pM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 1 pM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 0.01 nM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 0.1 nM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 1 nM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 0.01 pM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 0.1 pM. In some embodiments, the concentration of target RNA in a reverse transcription reaction mixture is about 1 pM.Reaction Mixture

[0074] The present disclosure further provides for reaction mixtures for reverse transcribing nucleic acid molecules, as well as reverse transcription methods employing such reaction solutions and product nucleic acid molecules produced using such methods, which comprise an anionic polymer for enhancing the activity of the polymerase, e.g., reverse transcriptase. In many instances, reaction mixtures described herein may contain one or more of the following components: (1) one or more buffering agent (e.g., sodium phosphate, sodium acetate, 2-(N-moropholino)-ethanesulfonic acid (MES), tris-(hydroxymethyl)aminomethane (Tris), 3 -(cyclohexylamino)-2-hydroxy-l -propanesulfonic acid (CAPS), citrate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), acetate, 3-(N- morpholino)prpoanesulfonic acid (MOPS), N-tris(hydroxymethyl)methyl-3- aminopropanesulfonio acid (TAPS), etc.), (2) one or more monovalent cationic salt (e.g., LiCl, NaCl, KC1, NH4CI, RbCl, CsCl, etc.), (3) one or more divalent cationic salt (e.g., MnCh, MgCh, MgSO4, CaCh, etc.), (4) one or more reducing agent (e.g., dithiothreitol, 2-mercaptoethanol, Tris (2-Carboxy ethyl) phosphine Hydrochloride, etc.), (5) one or more ioninc or non-ionic detergent (e.g., TRITON X-100™, NONIDET P40™, sodium dodecyl sulphate, etc.), (6) one or more crowding or stabilizing agents (e.g., trehalose, betaine, BSA, glycerol, PEG8000, etc.) (7) one or more DNA polymerase inhibitor (e.g., Actinomycin D, etc.), (8) nucleotides (e.g., dNTPs, such as dGTP, dATP, dCTP, dTTP, etc.), (9) RNAto be reverse transcribed and / or amplified, (10) one or more RNase inhibitor (e.g., RNASEOUT™, Invitrogen Corporation, Carlsbad, Calif, etc ), (11) a reverse transcriptase, and / or (12) one or more diluent (e.g., water). Other components and / or constituents (e.g., an oligonucleotide primer, e.g., an RT primer or TSO primer) may also be present in reaction mixtures described herein.

[0075] In some embodiments, the reaction mixture comprises an optimized reaction buffer that enhances the RT activity of a reverse transcriptase, e.g., UltraMarathonRT® or MarathonRT™. In some embodiments, the optimized reaction buffer comprises a buffering agent, e.g., sodium phosphate, sodium acetate, 2-(N-moropholino)-ethanesulfonic acid (MES), tris-(hydroxymethyl)aminomethane (Tris), 3-(cyclohexylamino)-2-hydroxy-l -propanesulfonic acid (CAPS), citrate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), acetate, 3- (N-morpholino)prpoanesulfonic acid (MOPS), N-tris(hydroxymethyl)methyl-3- aminopropanesulfonio acid (TAPS). In some embodiments, the buffering agent in the optimized reaction buffer is sodium phosphate. In some embodiments, the buffering agent in the optimized reaction buffer is sodium acetate. In some embodiments, the buffering agent in the optimized reaction buffer is 2-(N-moropholino)-ethanesulfonic acid (MES). In some embodiments, the buffering agent in the optimized reaction buffer is tris-(hydroxymethyl)aminomethane (Tris). In some embodiments, the buffering agent in the optimized reaction buffer is 3-(cyclohexylamino)- 2-hydroxy-l -propanesulfonic acid (CAPS). In some embodiments, the buffering agent in the optimized reaction buffer is N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). In some embodiments, the buffering agent in the optimized reaction buffer is acetate. In some embodiments, the buffering agent in the optimized reaction buffer is 3-(N-morpholino)prpoanesulfonic acid (MOPS). In some embodiments, the buffering agent in the optimized reaction buffer is N-tris(hydroxymethyl)methyl-3-aminopropanesulfonio acid (TAPS). In some embodiments, the optimized reaction buffer comprises a crowding or stabilizing agent, e.g., polyethylene glycol (PEG) or glycerol. In some embodiments, the crowding agent in the optimized reaction buffer is PEG. In some embodiments, the crowding agent in the optimized reaction buffer is PEG8000. In some embodiments, the crowding agent in the optimized reaction buffer is glycerol. In some embodiments, the optimized reaction buffer comprises a monovalent cationic salt, e.g., LiCl, NaCl, KC1, NH4Q, RbCl, CsCl. In some embodiments, the monovalent cationic salt in the optimized reaction buffer is LiCl. In some embodiments, the monovalent cationic salt in the optimized reaction buffer is KC1. In some embodiments, the monovalent cationic salt in the optimized reaction buffer is NH4CI. In some embodiments, the monovalent cationic salt in the optimized reaction buffer is RbCl. In some embodiments, the monovalent cationic salt in the optimized reaction buffer is CsCl. In some embodiments, the optimized reaction buffer comprises a divalent cationic salt, e.g., MnCk, MgCk, MgSCh, CaCk, SrCk, BaCk. In some embodiments, the divalent cationic salt in the optimized reaction buffer is MnCk. In some embodiments, the divalent cationic salt in the optimized reaction buffer is MgCk. In some embodiments, the divalent cationic salt in the optimized reaction buffer is MgSCU In some embodiments, the divalent cationic salt in the optimized reaction buffer is CaCk. In some embodiments, the divalent cationic salt in the optimized reaction buffer is SrCk. In some embodiments, the divalent cationic salt in the optimized reaction buffer is BaCk. In some embodiments, the optimized reaction buffer comprises a crowding agent, e.g., glycerol or PEG8000, at a concentration of about 1% to 50%. In some embodiments, the optimized reaction buffer comprises a buffering agent, e.g., MOPS, MES, HEPES, CAPS, TAPS, acetate, phosphate, or Tris, at a concentration of about 0.01 mM to about 1 M. In some embodiments, the optimized reaction buffer comprises a monovalent cationic salt, e.g., LiCl, NaCl, NH4Q, RbCl, CsCl or KC1, at a concentration of about 1 mM to about 1 M. In some embodiments, the optimized reaction buffer comprises a divalent cationic salt, e.g., MnCk, MgCk, MgSCh, CaCk, SrCk, BaCk, at a concentration of about 0.01 mM to about 100 mM. In some embodiments, the optimized reaction buffer comprises DTT at a concentration of about 0.1 mM to about 50 mM. In some embodiments, the pH of the optimized reaction buffer is about 6.5 to about 9. In some embodiments, the optimized reaction buffer comprises a crowding agent, e.g., glycerol orPEG8000, at a concentration of about 1% to 50%; a buffering agent, e.g., MOPS, MES, HEPES, CAPS, TAPS, acetate, phosphate, or Tris, at a concentration of about 0.01 mM to about 1 M; a monovalent cationic salt, e.g., LiCl, NaCl, NEUC1, RbCl, CsCl or KC1, at a concentration of about 1 mM to about 1 M; a divalent cationic salt, e.g., MnCh, MgCh, MgSO4, CaCh, SrCh, BaCh, at a concentration of about 0.01 mM to about 100 mM; and DTT at a concentration of about 0.1 mM to about 50 mM, and wherein the reaction buffer has a pH of about 6.5 to 9. In one embodiment, the optimized reaction buffer comprises PEG8000 at a concentration of about 1% to 20%, Tris at a concentration of about lOmM to about lOOmM; KC1 at a concentration of about 20mM to about 500mM, MgCh at a concentration of about O.lmM to about 20mM, and DTT at a concentration of about ImM to about lOmM, and wherein the reaction buffer has a pH of about 6.0 to 9.0. In one embodiment, the optimized reaction buffer comprises PEG8000 at a concentration of about 1% to 20%, MOPS at a concentration of about lOmM to about lOOmM; KC1 at a concentration of about 20mM to about 500mM, MgCh at a concentration of about 0. ImM to about 20mM, and DTT at a concentration of about ImM to about lOmM, and wherein the reaction buffer has a pH of about 6.0 to 9.0. In one embodiment, the optimized reaction buffer comprises PEG8000 at a concentration of about 1% to 20%, MES at a concentration of about lOmM to about lOOmM; KC1 at a concentration of about 20mM to about 500mM, MgCh at a concentration of about O.lmM to about 20mM, and DTT at a concentration of about ImM to about lOmM, and wherein the reaction buffer has a pH of about 6.0 to 9.0. In one embodiment, the optimized reaction buffer comprises PEG8000 at a concentration of about 1% to 20%, HEPES at a concentration of about lOmM to about lOOmM; KC1 at a concentration of about 20mM to about 500mM, MgCh at a concentration of about O.lmM to about 20mM, and DTT at a concentration of about ImM to about lOmM, and wherein the reaction buffer has a pH of about 6.0 to 9.0. In one embodiment, the optimized reaction buffer comprises glycerol at a concentration of about 1% to 40%, Tris at a concentration of about lOmM to about lOOmM; KC1 at a concentration of about 20mM to about 500mM, MgCh at a concentration of about O.lmM to about 20mM, and DTT at a concentration of about ImM to about lOmM, and wherein the reaction buffer has a pH of about 6.0 to 9.0. In one embodiment, the optimized reaction buffer comprises PEG8000 at a concentration of about 1% to 20%, MOPS at a concentration of about lOmM to about lOOmM; KC1 at a concentration of about 20mM to about 500mM, MgCh at a concentration of about O.lmM to about 20mM, and DTT at a concentration of about ImM toabout 1 OmM, and wherein the reaction buffer has a pH of about 6.0 to 9.0. In one embodiment, the optimized reaction buffer comprises glycerol at a concentration of about 1% to 40%, MES at a concentration of about lOmM to about lOOmM; KC1 at a concentration of about 20mM to about 500mM, MgCE at a concentration of about O.lmM to about 20mM, and DTT at a concentration of about ImM to about lOmM, and wherein the reaction buffer has a pH of about 6.0 to 9.0. In one embodiment, the optimized reaction buffer comprises glycerol at a concentration of about 1% to 40%, HEPES at a concentration of about lOmM to about lOOmM; KC1 at a concentration of about 20mM to about 500mM, MgCh at a concentration of about 0. ImM to about 20mM, and DTT at a concentration of about ImM to about lOmM, and wherein the reaction buffer has a pH of about 6.0 to 9.0. In one embodiment, the optimized reaction buffer comprises about 20% glycerol, about 50 mM Tris, about 200 mM KC1, about 2 mM MgCh, about 5 mM DTT; and has a pH of about 8.3.

[0076] In one embodiment, the optimized reaction buffer further comprises a protein stabilizing agent. Exemplary protein stabilizing agents include, but are not limited to, osmolytic stabilizers such as glycerol, erythritol, arabitol, sorbitol, mannitol, xylitol, mannisdomannitol, glucosylglycerol, glucose, fructose, sucrose, trehalose, isofluorosid, dextrans, levans, and polyethylene glycol; amino acids and derivatives thereof such as glycine, alanine, proline, taurine, betaine, octopine, glutamate, sarcosine, y-aminobutyric acid, trimethylamine, N-oxide (TMAO); ionic stabilizers such as citrate, sulfates, acetate, phosphates, and quaternary amines; and proteins such as bovine serum albumin (BSA).

[0077] In one embodiment, the optimized reaction buffer comprises trehalose at a concentration of about 0.1 M to about 2 M. In one embodiment, the optimized reaction buffer comprises betaine at a concentration of about 0.1 M to about 10 M. In one embodiment, the optimized reaction buffer comprises BSA at a concentration of about 0.5mg / mL to about 2mg / mL. In one embodiment, the optimized reaction buffer comprises glycerol at a concentration of about 1% to about 50%.

[0078] The concentration of the buffering agent in the reaction mixtures described herein may vary with the particular buffering agent used. Typically, the working concentration (i.e., the concentration in the reaction mixture) of the buffering agent will be from about 5 mM to about 500 mM (e.g., about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM,about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, from about 5 mM to about 500 mM, from about 10 mM to about 500 mM, from about 20 mM to about 500 mM, from about 25 mM to about 500 mM, from about 30 mM to about 500 mM, from about 40 mM to about 500 mM, from about 50 mM to about 500 mM, from about 75 mM to about 500 mM, from about 100 mM to about 500 mM, from about 25 mM to about 50 mM, from about 25 mM to about 75 mM, from about 25 mM to about 100 mM, from about 25 mM to about 200 mM, from about 25 mM to about 300 mM, etc.). When Tris (e.g., Tris-HCl) is used, the Tris working concentration will typically be from about 5 mM to about 100 mM, from about 5 mM to about 75 mM, from about 10 mM to about 75 mM, from about 10 mM to about 60 mM, from about 10 mM to about 50 mM, from about 25 mM to about 50 mM, etc.

[0079] The final pH of solutions of the invention will generally be set and maintained by buffering agents present in reaction solutions of the invention. The pH of reaction solutions of the invention, and hence reaction mixtures of the invention, will vary with the particular use and the buffering agent present but will often be from about pH 5.5 to about pH 9.0 (e.g., about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, about pH 7.4, about pH 7.5, about pH 7.6, about pH 7.7, about pH 7.8, about pH 7.9, about pH 8.0, about pH 8.1, about pH 8.2, about pH 8.3, about pH 8.4, about pH 8.5, about pH 8.6, about pH 8.7, about pH 8.8, about pH 8.9, about pH 9.0, from about pH 6.0 to about pH 8.5, from about pH 6.5 to about pH 8.5, from about pH 7.0 to about pH 8.5, from about pH 7.5 to about pH 8.5, from about pH 6.0 to about pH 8.0, from about pH 6.0 to about pH 7.7, from about pH 6.0 to about pH 7.5, from about pH 6.0 to about pH 7.0, from about pH 7.2 to about pH 7.7, from about pH 7.3 to about pH 7.7, from about pH 7.4 to about pH 7.6, from about pH 7.0 to about pH 7.4, from about pH 7.6 to about pH 8.0, from about pH 7.6 to about pH 8.5, from about pH 7.7 to about pH 8.5, from about pH 7.9 to about pH 8.5, from about pH 8.0 to about pH 8.5, from about pH 8.2 to about pH 8.5, from about pH 8.3 to about pH 8.5, from about pH 8.4 to about pH 8.5, from about pH 8.4 to about pH 9.0, from about pH 8.5 to about pH 9.0, etc.)

[0080] As indicated, one or more monovalent cationic salts (e.g., LiCl, NaCl, KC1, NH4CI, RbCl, CsCl, etc.) may be included in reaction solutions of the invention. In many instances, salts used in reaction solutions of the invention will dissociate in solution to generate at least one species which is monovalent (e.g., Li+, Na+, K+, NH4+, Rb+, Cs+etc.) When included in reaction solutions of the invention, salts will often be present either individually or in acombined concentration of from about 0.5 mM to about 500 mM (e.g., about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 10 mM, about 12 mM, about 15 mM, about 17 mM, about 20 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 27 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 64 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 120 mM, about 140 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400 mM, from about 1 mM to about 500 mM, from about 5 mM to about 500 mM, from about 10 mM to about 500 mM, from about 20 mM to about 500 mM, from about 30 mM to about 500 mM, from about 40 mM to about 500 mM, from about 50 mM to about 500 mM, from about 60 mM to about 500 mM, from about 65 mM to about 500 mM, from about 75 mM to about 500 mM, from about 85 mM to about 500 mM, from about 90 mM to about 500 mM, from about 100 mM to about 500 mM, from about 125 mM to about 500 mM, from about 150 mM to about 500 mM, from about 200 mM to about 500 mM, from about 10 mM to about 100 mM, from about 10 mM to about 75 mM, from about 10 mM to about 50 mM, from about 20 mM to about 200 mM, from about 20 mM to about 150 mM, from about 20 mM to about 125 mM, from about 20 mM to about 100 mM, from about 20 mM to about 80 mM, from about 20 mM to about 75 mM, from about 20 mM to about 60 mM, from about 20 mM to about 50 mM, from about 30 mM to about 500 mM, from about 30 mM to about 100 mM, from about 30 mM to about 70 mM, from about 30 mM to about 50 mM, etc ).

[0081] As indicated, one or more divalent cationic salts (e.g., MnCh, MgCk, MgSC , CaCb, etc.) may be included in reaction solutions of the invention. In many instances, salts used in reaction solutions of the invention will dissociate in solution to generate at least one species which is divalent (e.g., Mg++, Mn++, Ca++, etc.) When included in reaction solutions of the invention, salts will often be present either individually or in a combined concentration of from about 0.1 mM to about 500 mM (e.g., about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 12 mM, about 15 mM, about 17 mM, about 20 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 27 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 64 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85mM, about 90 M, about 95 mM, about 100 mM, about 120 mM, about 140 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400 mM, from about 1 mM to about 500 mM, from about 5 mM to about 500 mM, from about 10 mM to about 500 mM, from about 20 mM to about 500 mM, from about 30 mM to about 500 mM, from about 40 mM to about 500 mM, from about 50 mM to about 500 mM, from about 60 mM to about 500 mM, from about 65 mM to about 500 mM, from about 75 mM to about 500 mM, from about 85 mM to about 500 mM, from about 90 mM to about 500 mM, from about 100 mM to about 500 mM, from about 125 mM to about 500 mM, from about 150 mM to about 500 mM, from about 200 mM to about 500 mM, from about 10 mM to about 100 mM, from about 10 mM to about 75 mM, from about 10 mM to about 50 mM, from about 20 mM to about 200 mM, from about 20 mM to about 150 mM, from about 20 mM to about 125 mM, from about 20 mM to about 100 mM, from about 20 mM to about 80 mM, from about 20 mM to about 75 mM, from about 20 mM to about 60 mM, from about 20 mM to about 50 mM, from about 30 mM to about 500 mM, from about 30 mM to about 100 mM, from about 30 mM to about 70 mM, from about 30 mM to about 50 mM, etc.).

[0082] When included in reaction mixtures described herein, reducing agents (e.g., dithiothreitol, P-mercaptoethanol, Tris (2-Carboxyethyl) phosphine Hydrochloride, etc.) will often be present either individually or in a combined concentration of from about 0.1 mM to about 50 mM (e.g., about 0.2 mM, about 0.3 mM, about 0.5 mM, about 0.7 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 10 mM, about 12 mM, about 15 mM, about 17 mM, about 20 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 27 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, from about 0.1 mM to about 50 mM, from about 0.5 mM to about 50 mM, from about 1 mM to about 50 mM, from about 2 mM to about 50 mM, from about 3 mM to about 50 mM, from about 0.5 mM to about 20 mM, from about 0.5 mM to about 10 mM, from about 0.5 mM to about 5 mM, from about 0.5 mM to about 2.5 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 1 mM to about3.4 mM, from about 0.5 mM to about 3.0 mM, from about 1 mM to about 3.0 mM, from about1.5 mM to about 3.0 mM, from about 2 mM to about 3.0 mM, from about 0.5 mM to about 2.5 mM, from about 1 mM to about 2.5 mM, from about 1.5 mM to about 2.5 mM, from about 2 mM to about 3.0 mM, from about 2.5 mM to about 3.0 mM, from about 0.5 mM to about 2 mM, fromabout 0.5 mM to about 1.5 mM, from about 0.5 mM to about 1 .1 mM, from about 5.0 mM to about 10 mM, from about 5.0 mM to about 15 mM, from about 5.0 mM to about 20 mM, from about 10 mM to about 15 mM, from about 10 mM to about 20 mM, etc.).

[0083] Reaction mixtures described herein may also contain one or more ionic or nonionic detergent (e.g., TRITON X-100™, NONIDET P40™, sodium dodecyl sulfate, etc.). When included in reaction solutions of the invention, detergents will often be present either individually or in a combined concentration of from about 0.01% to about 5.0% (e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.3%, about 0.5%, about 0.7%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, from about 0.01% to about 5.0%, from about 0.01% to about 4.0%, from about 0.01% to about 3.0%, from about 0.01% to about 2.0%, from about 0.01% to about 1.0%, from about 0.05% to about 5.0%, from about 0.05% to about 3.0%, from about 0.05% to about 2.0%, from about 0.05% to about 1.0%, from about 0. 1% to about 5.0%, from about 0.1% to about 4.0%, from about 0.1% to about 3.0%, from about 0.1% to about 2.0%, from about 0.1% to about 1.0%, from about 0.1% to about 0.5%, etc.). For example, reaction solutions of the invention may contain TRITON X-100™ at a concentration of from about 0.01% to about 2.0%, from about 0.03% to about 1.0%, from about 0.04% to about 1.0%, from about 0.05% to about 0.5%, from about 0.04% to about 0.6%, from about 0.04% to about 0.3%, etc.

[0084] Reaction mixtures described herein may also contain one or more stabilizing agents (e.g., PEG8000, trehalose, betaine, BSA, glycerol, etc.). In some embodiments, when included in reaction solutions of the invention, stabilizing agents are present either individually or in a combined concentration from 0.01 M to about 50 M (e.g., about 0.05M, about 0.1 M, 0.2 M, about 0.3 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.9 M, about 1 M, about 2 M, about 3 M, about 4 M, about 5 M, about 6 M, about 10 M, about 12 M, about 15 M, about 17 M, about 20 M, about 22 M, about 23 M, about 24 M, about 25 M, about 27 M, about 30 M, about 35 M, about 40 M, about 45 M, about 50 M, from about 0.1 M to about 1 M, from about 0.5 M to about 5 M, from about 0.2 M to about 2 M, from about 0.3 M to about 3 M, from about 0.4 M to about 4 M, from about 0.5 M to about 5 M, from about 0.2 M to about 0.8 M, from about 0.5 M to about 1 M, from about 0.05 M to about 1 M, from about 0.05 M to about 10 M, from about 0.05 M to about 20M, etc.). In some embodiments, when included in reaction mixtures describedherein, such stabilizing agents are present either individually or in a combined concentration of from about 0.01 mg / ml to about 100 mg / ml (e.g., about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.1 mg / ml, about 0.11 mg / ml, about 0.12 mg / ml, about 0.15 mg / ml, about 0.17 mg / ml, about 0.2 mg / ml, about 0.25 mg / ml, about 0.35 mg / ml, about 0.5 mg / ml, about 0.75mg / ml, about 1.0 mg / ml, about 1.5 mg / ml, about 2.0 mg / ml, about 2.5 mg / ml, about 3.0 mg / ml, about 3.5 mg / ml, about 4.0 mg / ml, about 5.0 mg / ml, about 6.0 mg / ml, about 7.0 mg / ml, about 8.0 mg / ml, about 9.0 mg / ml, about 10.0 mg / ml, from about 0.05 mg / ml to about 3.0 mg / ml, from about 0.1 mg / ml to about 5.0 mg / ml, from about 0.2 mg / ml to about 2.0 mg / ml, etc.). In some embodiments, when included in reaction mixtures described herein, such stabilizing agents are be present either individually or in a combined concentration of from about 0.1% to about 50% (e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 5.0%, about 7.0%, about 9.0%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 22%, about 25%, about 27%, about 30%, about 35%, about 40%, about 45%, about 50%, from about 0.1% to about 50%, from about 0.1% to about 40%, from about 0.1% to about 30%, from about 0.0% to about 20%, from about 0.1% to about 10%, etc.

[0085] Reaction mixtures described herein may also contain one or more DNA polymerase inhibitor (e.g., Actinomycin D, etc.). When included in reaction solutions of the invention, such inhibitors will often be present either individually or in a combined concentration of from about 0.1 pg / ml to about 100 pg / ml (e.g., about 0.1 pg / ml, about 0.2 pg / ml, about 0.3 pg / ml, about 0.4 pg / ml, about 0.5 pg / ml, about 0.6 pg / ml, about 0.7 pg / ml, about 0.8 pg / ml, about 0.9 pg / ml, about 1.0 pg / ml, about 1.1 pg / ml, about 1.3 pg / ml, about 1.5 pg / ml, about 1.7 pg / ml, about 2.0 pg / ml, about 2.5 pg / ml, about 3.5 pg / ml, about 5.0 pg / ml, about 7.5 pg / ml, about 10 pg / ml, about 15 pg / ml, about 20 pg / ml, about 25 pg / ml, about 30 pg / ml, about 35 pg / ml, about 40 pg / ml, about 50 pg / ml, about 60 pg / ml, about 70 pg / ml, about 80 pg / ml, about 90 pg / ml, about 100 pg / ml, from about 0.5 pg / ml to about 30 pg / ml, from about 0.75 pg / ml to about 30 pg / ml, from about 1.0 pg / ml to about 30 pg / ml, from about 2.0 pg / ml to about 30 pg / ml, from about 3.0 pg / ml to about 30 pg / ml, from about 4.0 pg / ml to about 30 pg / ml, from about 5.0 pg / ml to about 30 pg / ml, from about 7.5 pg / ml to about 30 pg / ml, from about 10 pg / ml to about 30 pg / ml, from about 15 pg / ml to about 30 pg / ml, from about 0.5 pg / ml to about 20[ig / ml, from about 0.5 [ig / ml to about 10 pg / ml, from about 0.5 pg / ml to about 5 pg / ml, from about 0.5 .g / ml to about 2 [ig / ml, from about 0.5 [ g / ml to about 1 [ g / ml, from about 1 [ g / ml to about 10 [ g / ml, from about 1 [ig / ml to about 5 [ig / ml, from about 1 pg / ml to about 2 [ig / ml, from about 1 [ig / ml to about 100 [ig / ml, from about 10 [ig / ml to about 100 [ig / ml, from about 20 [ig / ml to about 100 [ig / ml, from about 40 [ig / ml to about 100 [ig / ml, from about 30 [ig / ml to about 80 [ig / ml, from about 30 |ig / ml to about 70 [ig / ml, from about 40 [ig / ml to about 60 [ig / ml, from about 40 [ig / ml to about 70 [ig / ml, from about 40 [ig / ml to about 80 [ig / ml, etc.).

[0086] Reaction mixtures described herein may also contain one or more additional additives that improve reverse transcription activity, including agents that improve primer utilization efficiency and improve product yield. In one embodiment, the reaction solution comprises an agent that reduces non-specific binding of primers to the MarathonRT™ surface. The agent may comprise any protein, nucleic acid molecule, or small molecule that prevents or reduces non-specific binding. In certain embodiments, the agent comprises D4ARNA or variant thereof. D4A and variants of D4 A that can be included in the reverse transcription assay of the invention include, but are not limited to, those described in detail in International Patent Publication W02019005955A1, which is incorporated by reference herein in its entirety.

[0087] When included in reaction mixtures described herein, D4A, or variant thereof, may be present at ratio of D4A (or variant thereof) concentration to MarathonRT™ concentration from about 0.1 : 1 to about 100: 1. For example, in some embodiments, D4A, or variant thereof, may be present at ratio of D4A (or variant thereof) concentration to MarathonRT™ concentration of about 0.1 : 1, 0.2: 1, 0.3: 1, 0.4:1, 0.5: 1, 0.6:1, 0.7:1, 0.8:1, 0.9: 1, 1 :1, 2: 1, 3: 1, 4: 1, 5: 1, 6:1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16:1, 17: 1, 18: 1, 19: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40:1, 45: 1, 50: 1, 55: 1, 60: 1, 65:1, 70:1, 75: 1, 80: 1, 85: 1, 90: 1, 95: 1, or 100:1.

[0088] In some embodiments, the additive that improves reverse transcription activity is an anionic polymer. An anionic polymer, as described herein, is any naturally occurring or non- naturally occurring polymer, e.g., a plurality of repeating monomer units bearing an overall negative charge. The anionic polymer may comprise a homogenous set of monomeric units, or may comprise a heterogenous set of monomeric units, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different monomeric units. The anionic polymer may be a linear polymer, branched polymer, or cross-linked polymer. In some embodiments, the anionic polymer is naturally occurring. In some embodiments, the anionic polymer comprises an oligonucleotide, peptide,polypeptide, or oligosaccharide, each of which independently bears a net negative charge. Tn some embodiments, the anionic polymer is non-naturally occurring. In some embodiments, the anionic polymer is an oligonucleotide, e.g., a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA), e.g., carrier RNA. In some embodiments, the anionic polymer is DNA. In some embodiments, the anionic polymer is RNA. In some embodiments, the anionic polymer is a peptide. In some embodiments, the anionic polymer is a polypeptide. In some embodiments, the anionic polymer is an oligosaccharide, e.g., a glycosaminoglycan, e.g., alginate, hyaluronate, dextran, or heparin. In some embodiments, the anionic polymer is a glycosaminoglycan. In some embodiments, the anionic polymer is alginate. In some embodiments, the anionic polymer is hyaluronate. In some embodiments, the anionic polymer is heparin. In other embodiments, the anionic polymer is non-naturally occurring. The anionic polymer may comprise polystyrene, polyethylene, polypropylene, polyacetylene, poly(vinyl chloride) (PVC), polyolefin copolymers, poly(urethane)s, polyacrylic acid (PAA), polymethacrylates, polyacrylamides and polymethacrylamides, poly(methyl methacrylate), poly(2-hydroxyethyl methacrylate), polyesters, polysiloxanes, polydimethylsiloxane (PDMS), polyethers, poly(orthoester), poly(carbonates), poly(hydroxyalkanoate)s, polyfluorocarbons, polyethylene glycol, nylon, polyalkenes, phenolic resins, natural and synthetic elastomers, adhesives and sealants, polyolefins, polysulfones, polyacrylonitrile, poly(glycolic acid), poly(lactic acid) (PLA), poly(lactic glycolic acid) (PLGA), a polydioxanone (PDA), polycarbonates, (e g., polyamides (e.g., nylon)), fluoroplastics, carbon fiber, and blends or copolymers thereof. In an embodiment, the anionic polymer comprises PLA or PLGA. In an embodiment, the anionic polymer comprises PAA.

[0089] In many instances, nucleotides (e.g., dNTPs, such as dGTP, dATP, dCTP, dTTP, etc.) will be present in reaction mixtures described herein. Typically, individual nucleotides will be present in concentrations of from about 0.001 mM to about 50 mM (e.g., about 0.001 mM, 0.01 mM, 0.07 mM, about 0.1 mM, about 0.15 mM, about 0.18 mM, about 0.2 mM, about 0.3 mM, about 0.5 mM, about 0.7 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 10 mM, about 12 mM, about 15 mM, about 17 mM, about 20 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 27 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, from about 0.1 mM to about 50 mM, from about 0.5 mM to about 50 mM, from about 1 mM to about 50 mM, from about 2 mMto about 50 mM, from about 3 mM to about 50 mM, from about 0.5 mM to about 20 mM, from about 0.5 mM to about 10 mM, from about 0.5 mM to about 5 mM, from about 0.5 mM to about 2.5 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 1 mM to about 3.4 mM, from about 0.5 mM to about 3.0 mM, from about 1 mM to about 3.0 mM, from about 1.5 mM to about 3.0 mM, from about 2 mM to about 3.0 mM, from about 0.5 mM to about 2.5 mM, from about 1 mM to about 2.5 mM, from about 1.5 mM to about 2.5 mM, from about 2 mM to about 3.0 mM, from about 2.5 mM to about 3.0 mM, from about 0.5 mM to about 2 mM, from about 0.5 mM to about 1.5 mM, from about 0.5 mM to about 1.1 mM, from about 5.0 mM to about 10 mM, from about 5.0 mM to about 15 mM, from about 5.0 mM to about 20 mM, from about 10 mM to about 15 mM, from about 10 mM to about 20 mM, etc.). The combined nucleotide concentration, when more than one nucleotide is present, can be determined by adding the concentrations of the individual nucleotides together. When more than one nucleotide is present in reaction mixtures described herein, the individual nucleotides may not be present in equimolar amounts. Thus, a reaction solution may contain, for example, 1 mM dGTP, 1 mM dATP, 0.5 mM dCTP, and 1 mM dTTP

[0090] RNA will typically be present in reaction mixtures described herein. In most instances, RNA will be added to the reaction mixture shortly prior to reverse transcription. Thus, reaction solutions may be provided without RNA. This will typically be the case when reaction solutions are provided in kits. RNA, when present in reaction solutions will often be present in a concentration of 0.01 picogram to 100 pg / 20 pl reaction mixture (e.g., about 0.01 picogram / 20 pl, about 0.1 picogram / 20 pl, about 0.5 picogram / 20 pl, about 1 picogram / 20 pl, about 10 picograms / 20 pl, about 50 picograms / 20 pl, about 100 picograms / 20 pl, about 200 picograms / 20 pl, about 10 picograms / 20 pl, about 500 picograms / 20 pl, about 800 picograms / 20 pl, about 1.0 nanogram / 20 pl, about 5.0 nanograms / 20 pl, about 10 nanograms / 20 pl, about 25 nanograms / 20 pl, about 50 nanograms / 20 pl, about 75 nanograms / 20 pl, about 100 nanograms / 20 pl, about 150 nanograms / 20 pl, about 250 nanograms / 20 pl, about 400 nanograms / 20 pl, about 500 nanograms / 20 pl, about 750 nanograms / 20 pl, about 1.0 pg / 20 pl, about 5.0 pg / 20 pl, about 10 pg / 20 pl, about 20 pg / 20 pl, about 30 pg / 20 pl, about 40 pg / 20 pl, about 50 pg / 20 pl, about 70 pg / 20 pl, about 85 pg / 20 pl, about 100 pg / 20 pl, from about 10 picograms / 20 pl to about 100 pg / 20 pl, from about 10 picograms / 20 pl to about 100 pg / 20 pl, from about 100 picograms / 20 pl to about 100 pg / 20 pl, from about 1.0 nanograms / 20 pl to about 100 pg / 20 pl, from about 100nanograms / 20 gl to about 100 gg / 20 gl, from about 10 picograms / 20 pl to about 10 gg / 20 gl, from about 10 picograms / 20 pl to about 5 gg / 20 gl, from about 100 nanograms / 20 gl to about 5 gg / 20 gl, from about 1 pg / 20 gl to about 10 gg / 20 gl, from about 1 pg / 20 gl to about 5 gg / 20 gl, from about 100 nanograms / 20 gl to about 1 gg / 20 gl, from about 500 nanograms / 20 gl to about 5 gg / 20 gl, etc.). As one skilled in the art would recognize, different reverse transcription reactions may be performed in volumes other than 20 gl. In such instances, the total amount of RNA present will vary with the volume used. Thus, the above amounts are provided as examples of the amount of RNA / 20 gl of reaction solution.

[0091] A polymerase, e.g., a reverse transcriptase, may also be present in reaction mixtures described herein. When present, the polymerase, e.g., reverse transcriptase, will often be present in a concentration which results in about 0.01 to about 1,000 units of reverse transcriptase activity / gl (e.g., about 0.01 unit / gl, about 0.05 unit / gl, about 0.1 unit / gl, about 0.2 unit / gl, about 0.3 unit / gl, about 0.4 unit / gl, about 0.5 unit / gl, about 0.7 unit / gl, about 1.0 unit / gl, about 1.5 unit / gl, about 2.0 unit / gl, about 2.5 unit / gl, about 5.0 unit / gl, about 7.5 unit / gl, about 10 unit / gl, about 20 unit / gl, about 25 unit / gl, about 50 unit / gl, about 100 unit / gl, about 150 unit / gl, about 200 unit / gl, about 250 unit / gl, about 350 unit / gl, about 500 unit / gl, about 750 unit / gl, about 1,000 unit / gl, from about 0.1 unit / gl to about 1,000 unit / gl, from about 0.2 unit / gl to about 1,000 unit / gl, from about 1.0 unit / gl to about 1,000 unit / gl, from about 5.0 unit / gl to about 1,000 unit / gl, from about 10 unit / gl to about 1,000 unit / gl, from about 20 unit / gl to about 1,000 unit / gl, from about 50 unit / gl to about 1,000 unit / gl, from about 100 unit / gl to about 1,000 unit / gl, from about 200 unit / gl to about 1,000 unit / gl, from about 400 unit / gl to about 1,000 unit / gl, from about 500 unit / gl to about 1,000 unit / gl, from about 0.1 unit / gl to about 300 unit / gl, from about 0.1 unit / gl to about 200 unit / gl, from about 0.1 unit / gl to about 100 unit / gl, from about 0.1 unit / gl to about 50 unit / gl, from about 0.1 unit / gl to about 10 unit / gl, from about 0.1 unit / gl to about 5.0 unit / gl, from about 0.1 unit / gl to about 1.0 unit / gl, from about 0.2 unit / gl to about 0.5 unit / gl, etc. In certain embodiments, the reaction solution comprises a lower concentration of the reverse transcriptase described herein, as compared to what would be necessary to produce equivalent product from other reverse transcriptases.

[0092] Reaction mixtures described herein may be prepared as concentrated solutions (e.g., 5* solutions) which are diluted to a working concentration for final use. With respect to a 5* reaction solution, a 5: 1 dilution is required to bring such a 5* solution to a workingconcentration. Reaction mixtures described herein may be prepared, for examples, as a 2*, a 3xpa 4x, a 5x, a 6x, a 7x, a 8x, a 9x, a 10x, etc. solutions. One major limitation on the fold concentration of such solutions is that, when compounds reach particular concentrations in solution, precipitation occurs. Thus, concentrated reaction solutions will generally be prepared such that the concentrations of the various components are low enough so that precipitation of buffer components will not occur. As one skilled in the art would recognize, the upper limit of concentration which is feasible for each solution will vary with the particular solution and the components present.

[0093] In many instances, reaction mixtures described herein will be provided in sterile form. Sterilization may be performed on the individual components of reaction solutions prior to mixing or on reaction solutions after they are prepared. Sterilization of such solutions may be performed by any suitable means including autoclaving or ultrafdtration.Two-Step Method of Reverse Transcription and Template Switching

[0094] In one aspect, the present disclosure relates to a two-step method for reverse transcription and template switching. For example, the present disclosure provides a method for performing a first reverse transcription reaction, e.g., preparing a first reverse transcription reaction mixture, followed by a second template switching reaction, e.g., preparing a second template switching reaction mixture. In some embodiments, the method comprises preparing a first reverse transcription reaction mixture, e.g., a reaction mixture comprising the components for a reverse transcription reaction, e.g., a polymerase having reverse transcription activity and a target polynucleotide. In some embodiments, the method comprises preparing a second template switching reaction mixture, e.g., a reaction mixture comprising components for a template switching reaction, e.g., a polymerase having template switching activity and a template switching oligonucleotide (TSO). In some embodiments, the two-step method for reverse transcription and template switching comprises: (i) preparing a first reverse transcription reaction mixture, e.g., a reaction mixture comprising the components for a reverse transcription reaction; (ii) incubating the first reverse transcription reaction mixture for a period of time, e.g., a period of time sufficient for a target polynucleotide to be reverse transcribed; (iii) preparing a second template switching reaction mixture, e.g., a reaction mixture comprising the components for a template switching reaction; and (iv) incubating the template switching reaction mixture for aperiod of time, e.g., a period of time sufficient for template switching. In some embodiments, the two-step method for reverse transcription and template switching comprises preparing a first reverse transcription reaction mixture comprising the components for a reverse transcription reaction. In some embodiments, the two-step method for reverse transcription and template switching comprises incubating the first reverse transcription reaction mixture for a period of time sufficient for a target polynucleotide to be reverse transcribed. In some embodiments, the two-step method for reverse transcription and template switching comprises preparing a second template switching reaction mixture comprising the components for a template switching reaction. In some embodiments, the two-step method for reverse transcription and template switching comprises incubating the template switching reaction mixture for a period of time sufficient for template switching. In some embodiments, the reverse transcription reaction and template switching reaction are performed separately, e.g., as two individual steps. In some embodiments, the reverse transcription reaction is performed before the template switching reaction. In some embodiments, the template switching reaction is performed after the reverse transcription reaction. irst step reaction mixture

[0095] In one aspect, the two-step method for reverse transcription and template switching comprises a first reaction mixture, e.g., a reverse transcription reaction mixture. For example, the first reaction mixture comprises components capable of performing a reverse transcription reaction. In some embodiments, the reverse transcription reaction mixture is prepared separately from the template switching reaction mixture. In some embodiments, the reverse transcription reaction mixture comprises: a target RNA, e.g., a plurality of target RNAs, e.g., total cellular RNA, e.g., messenger RNA (mRNA), deoxyribonucleotide triphosphates (dNTPs), a polymerase, e.g., a reverse transcriptase, an oligonucleotide primer, e.g., a reverse transcription primer, carrier RNA, nuclease-free water, and a buffer. In some embodiments, the reverse transcription reaction mixture comprises a target RNA, dNTPs, a reverse transcription primer, carrier RNA, a buffer, and a group II intron reverse transcriptase, e.g., MarathonRT™ or UltraMarathonRT®. In some embodiments, the reverse transcription reaction mixture comprises a target RNA, dNTPs, an oligonucleotide primer, carrier RNA, a buffer, and MarathonRT™. In some embodiments, the reverse transcription reaction mixture comprises a target RNA, dNTPs,an oligonucleotide primer, carrier RNA, a buffer, and UltraMarathonRT®. In some embodiments, the reverse transcription reaction mixture comprises a target RNA, dNTPs, an oligonucleotide primer, a buffer, and MarathonRT™. In some embodiments, the reverse transcription reaction mixture comprises a target RNA, dNTPs, an oligonucleotide primer, a buffer, and UltraMarathonRT®.

[0096] In some embodiments, the reverse transcription reaction mixture comprises target RNA, e.g., total cellular RNA, e.g., mRNA. In some embodiments, the reverse transcription reaction mixture comprises total cellular RNA. In some embodiments, the reverse transcription reaction mixture comprises mRNA. In some embodiments, the reverse transcription reaction mixture comprises 1 ng to20 ng of total cellular RNA. In some embodiments, the reverse transcription reaction mixture comprises 0.1 ng to 20 ng of mRNA.

[0097] In some embodiments, the reverse transcription reaction mixture comprises deoxyribonucleotide triphosphates (dNTPs), e.g., deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), or deoxycytidine triphosphate (dCTP). In some embodiments, the reverse transcription reaction mixture comprises dATP. In some embodiments, the reverse transcription reaction mixture comprises dGTP. In some embodiments, the reverse transcription reaction mixture comprises dTTP. In some embodiments, the reverse transcription reaction mixture comprises dCTP. In some embodiments, the reverse transcription reaction mixture comprises a mixture of dATP, dTTP, dGTP, and dCTP. In some embodiments, the reverse transcription reaction mixture comprises a mixture of dATP, dTTP, dGTP, and dCTP wherein each dNTP is present in an equal amount. In some embodiments, the reverse transcription reaction mixture comprises an equimolar dNTP mixture at a concentration of about 0.1 mM to about 10 mM, e.g., about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, or 10 mM. In some embodiments, the reverse transcription reaction mixture comprises an equimolar dNTP mixture at a concentration of about 0. 1 mM. In some embodiments, the reverse transcription reaction mixture comprises an equimolar dNTP mixture at a concentration of about 0.5 mM. In some embodiments, the reverse transcription reaction mixture comprises an equimolar dNTP mixture at a concentration of about 1 mM. In some embodiments, the reverse transcription reaction mixture comprises an equimolar dNTP mixture at a concentration of about 2 mM. In some embodiments, the reverse transcription reaction mixture comprises an equimolar dNTP mixture at a concentration of about 5 mM. In some embodiments, the reverse transcriptionreaction mixture comprises an equimolar dNTP mixture at a concentration of about 10 mM. Tn some embodiments, the reverse transcription reaction mixture comprises an equimolar dNTP mixture at a concentration of 1 mM.

[0098] In some embodiments, the reverse transcription reaction mixture comprises an oligonucleotide primer, e.g., a reverse transcription primer. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcription primer. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcription primer at a concentration of about 0.05 pM to about 5 pM, e.g., about 0.05 pM, 0.1 pM, 0.5 pM, 1 pM, 2 pM, or 5 pM. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcription primer at a concentration of about 0.05 pM. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcription primer at a concentration of about 0.1 pM. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcription primer at a concentration of about 0.5 pM. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcription primer at a concentration of about 1 pM. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcription primer at a concentration of about 2 pM. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcription primer at a concentration of about 5 pM. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcription primer at a concentration of 0.5 pM.

[0099] In some embodiments, the reverse transcription reaction mixture comprises carrier RNA. In some embodiments, the reverse transcription reaction mixture comprises carrier RNA at a concentration of about 1 ng / pL to about 100 ng / pL, e.g., about 1 ng / pL, 5 ng / pL, 10 ng / pL, 25 ng / pL, 50 ng / pL, 75 ng / pL, or 100 ng / pL. In some embodiments, the reverse transcription reaction mixture comprises carrier RNA at a concentration of about 1 ng / pL. In some embodiments, the reverse transcription reaction mixture comprises carrier RNA at a concentration of about 5 ng / pL. In some embodiments, the reverse transcription reaction mixture comprises carrier RNA at a concentration of about 10 ng / pL. In some embodiments, the reverse transcription reaction mixture comprises carrier RNA at a concentration of about 25 ng / pL. In some embodiments, the reverse transcription reaction mixture comprises carrier RNA at a concentration of about 50 ng / pL. In some embodiments, the reverse transcription reaction mixture comprises carrier RNA at a concentration of about 75 ng / pL. In some embodiments, thereverse transcription reaction mixture comprises carrier RNA at a concentration of about 100 ng / pL. In some embodiments, the reverse transcription reaction mixture comprises carrier RNA at a concentration of 10 ng / pL.

[0100] In some embodiments, the reverse transcription reaction mixture comprises an RNase inhibitor, e.g., RNaseOUT™. In some embodiments, the reverse transcription reaction mixture comprises RNaseOUT™. In some embodiments, the reverse transcription reaction mixture comprises RNaseOUT™ at a concentration of about 1 unit / pL to about 200 units / pL, e.g., about 1 unit / pL, 2 units / pL, 5 units / pL, 10 units / pL, 50 units / pL, 100 units / pL, or 200 units / pL. In some embodiments, the reverse transcription reaction mixture comprises RNaseOUT™ at a concentration of about 1 unit / pL. In some embodiments, the reverse transcription reaction mixture comprises RNaseOUT™ at a concentration of about 2 units / pL. In some embodiments, the reverse transcription reaction mixture comprises RNaseOUT™ at a concentration of about 5 units / pL. In some embodiments, the reverse transcription reaction mixture comprises RNaseOUT™ at a concentration of about 10 units / pL. In some embodiments, the reverse transcription reaction mixture comprises RNaseOUT™ at a concentration of about 50 units / pL. In some embodiments, the reverse transcription reaction mixture comprises RNaseOUT™ at a concentration of about 100 units / pL. In some embodiments, the reverse transcription reaction mixture comprises RNaseOUT™ at a concentration of about 200 units / pL. In some embodiments, the reverse transcription reaction mixture comprises RNaseOUT™ at a concentration of 40 units / pL.

[0101] In some embodiments, the reverse transcription reaction mixture comprises a buffer. In some embodiments, the reverse transcription reaction mixture comprises a buffering agent, e.g., Tris-HCl. In some embodiments, the reverse transcription reaction mixture comprises Tris-HCl. In some embodiments, the reverse transcription reaction mixture comprises Tris-HCl at a concentration of about 5 mM to about 500 mM, e.g., about 5 mM, 10 mM, 50 mM, 100 mM, 250 mM, or 500 mM. In some embodiments, the reverse transcription reaction mixture comprises Tris-HCl at a concentration of about 5 mM. In some embodiments, the reverse transcription reaction mixture comprises Tris-HCl at a concentration of about 10 mM. In some embodiments, the reverse transcription reaction mixture comprises Tris-HCl at a concentration of about 50 mM. In some embodiments, the reverse transcription reaction mixture comprises Tris- HCl at a concentration of about 100 mM. In some embodiments, the reverse transcriptionreaction mixture comprises Tris-HCl at a concentration of about 250 mM. In some embodiments, the reverse transcription reaction mixture comprises Tris-HCl at a concentration of about 500 mM. In some embodiments, the reverse transcription reaction mixture comprises a salt solution, e.g., KC1 or MgCh. In some embodiments, the reverse transcription reaction mixture comprises KC1. In some embodiments, the reverse transcription reaction mixture comprises KC1 at a concentration of about 20 mM to about IM, e.g., about 20 mM, 50 mM, 100 mM, 200 mM, 500 mM, or IM. In some embodiments, the reverse transcription reaction mixture comprises KC1 at a concentration of about 20 mM. In some embodiments, the reverse transcription reaction mixture comprises KC1 at a concentration of about 50 mM. In some embodiments, the reverse transcription reaction mixture comprises KC1 at a concentration of about 100 mM. In some embodiments, the reverse transcription reaction mixture comprises KC1 at a concentration of about 200 mM. In some embodiments, the reverse transcription reaction mixture comprises KC1 at a concentration of about 500 mM. In some embodiments, the reverse transcription reaction mixture comprises KC1 at a concentration of about IM. In some embodiments, the reverse transcription reaction mixture comprises MgCh. In some embodiments, the reverse transcription reaction mixture comprises MgCh at a concentration of about 0.1 mM to about 50 mM, e.g., about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 25 mM, or 50 mM. In some embodiments, the reverse transcription reaction mixture comprises MgCh at a concentration of about 0.1 mM. In some embodiments, the reverse transcription reaction mixture comprises MgCh at a concentration of about 0.5 mM. In some embodiments, the reverse transcription reaction mixture comprises MgCh at a concentration of about 1 mM. In some embodiments, the reverse transcription reaction mixture comprises MgCh at a concentration of about 5 mM. In some embodiments, the reverse transcription reaction mixture comprises MgCh at a concentration of about 10 mM. In some embodiments, the reverse transcription reaction mixture comprises MgCh at a concentration of about 25 mM. In some embodiments, the reverse transcription reaction mixture comprises MgCh at a concentration of about 50 mM. In some embodiments, the reverse transcription reaction mixture comprises a reducing agent, e.g., dithiothreitol (DTT). In some embodiments, the reverse transcription reaction mixture comprises DTT. In some embodiments, the reverse transcription reaction mixture comprises DTT at a concentration of about 0.5 mM to about 50 mM, e.g., about 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 25 mM, or 50 mM. In some embodiments, the reverse transcription reaction mixture comprises DTT at a concentration ofabout 0.5 mM. In some embodiments, the reverse transcription reaction mixture comprises DTT at a concentration of about 1 mM. In some embodiments, the reverse transcription reaction mixture comprises DTT at a concentration of about 2 mM. In some embodiments, the reverse transcription reaction mixture comprises DTT at a concentration of about 5 mM. In some embodiments, the reverse transcription reaction mixture comprises DTT at a concentration of about 10 mM. In some embodiments, the reverse transcription reaction mixture comprises DTT at a concentration of about 25 mM. In some embodiments, the reverse transcription reaction mixture comprises DTT at a concentration of about 50 mM. In some embodiments, the reverse transcription reaction mixture comprises a crowding or stabilizing reagent, e.g. glycerol. In some embodiments, the reverse transcription reaction mixture comprises glycerol. In some embodiments, the reverse transcription reaction mixture comprises glycerol at a concentration of about 1% (vol / vol) to about 50% (vol / vol), e.g., about 1%, 5%, 10%, 25%, or 50%. In some embodiments, the reverse transcription reaction mixture comprises glycerol at a concentration of about 1%. In some embodiments, the reverse transcription reaction mixture comprises glycerol at a concentration of about 5%. In some embodiments, the reverse transcription reaction mixture comprises glycerol at a concentration of about 10%. In some embodiments, the reverse transcription reaction mixture comprises glycerol at a concentration of about 25%. In some embodiments, the reverse transcription reaction mixture comprises glycerol at a concentration of about 50%. In some embodiments, the reverse transcription reaction buffer comprises 50 mM of Tris-HCl pH 8.3, 200 mM of KC1, 4 mM of MgCh, 5 mM of DTT, and 20% glycerol. In some embodiments, the reverse transcription reaction mixture has greater ionic strength than the template switching reaction mixture.

[0102] In some embodiments, the reverse transcription reaction mixture comprises a polymerase, e.g., a DNA polymerase or a reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises a DNA polymerase. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcriptase, e.g., an MMLV reverse transcriptase or a group II intron reverse transcriptase. In some embodiments, the reverse transcriptase is an MMLV reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises SuperScript™, e.g., SuperScript™, SuperScript™ II, SuperScript™ III, or SuperScript™ IV. In some embodiments, the reverse transcription reaction mixturecomprises a group II intron reverse transcriptase, e.g., MarathonRT™ or UltraMarathonRT®. In some embodiments, the reverse transcription reaction mixture comprises MarathonRT™ reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises UltraMarathonRT® reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises Induro® RT. In some embodiments, the reverse transcription reaction mixture comprises TGIRT™. In some embodiments, the reverse transcription reaction mixture comprises about 0.1 units to about 100 units of a polymerase, e.g., reverse transcriptase, e.g., about 0.1, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 1,000 or more units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 0.1-1,000 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 1-900 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 10-800 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 100-700 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 200-600 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 300-500 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 500-1,000 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 1-500 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 10-200 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 100-200 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 1-20 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 1-15 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 1-10 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 1-5 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises between about 5-10 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 0.1 units of reverse transcriptase. In someembodiments, the reverse transcription reaction mixture comprises about 1 unit of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 2 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 5 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 10 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 20 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 30 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 40 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 50 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 60 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 70 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 80 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 90 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 100 units of reverse transcriptase. In some embodiments, the reverse transcription reaction mixture comprises about 10 units of MarathonRT™. In some embodiments, the reverse transcription reaction mixture comprises about 10 units of UltraMarathonRT®. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcriptase at a concentration of about 0.1 units / pL to about 20 units / pL, e.g., about 0.1 units / pL, 0.5 units / pL, 1 unit / pL, 2 units / pL, 5 units / pL, 10 units / pL, or 20 units / pL. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcriptase at a concentration of about 0.1 units / pL. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcriptase at a concentration of about 0.5 units / pL. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcriptase at a concentration of about 1 unit / pL. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcriptase at a concentration of about 2 units / pL. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcriptase at a concentration of about 5 units / pL. In some embodiments, the reverse transcription reaction mixture comprises a reverse transcriptase at a concentration of about 10 units / pL. In some embodiments, the reverse transcription reaction mixture comprises a reversetranscriptase at a concentration of about 20 units / pL. In some embodiments, the reverse transcription reaction mixture comprises UltraMarathonRT® at a concentration of 1 unit / pL.Second step reaction mixture

[0103] In one aspect, the two-step method for reverse transcription and template switching comprises a second reaction mixture, e.g., a template switching reaction mixture. For example, the second reaction mixture comprises components capable of performing a template switching reaction. In some embodiments, the second reaction mixture comprises the first reaction mixture, e.g., the template switching reaction mixture comprises the reverse transcription reaction mixture, e.g., the template switching reaction components are added to the reverse transcription reaction mixture. In some embodiments, the template switching reaction components are added to the reverse transcription reaction mixture to generate a template switching reaction mixture. In some embodiments, the template switching reaction mixture comprises in addition to the reverse transcription reaction mixture: a template switching oligonucleotide (TSO), deoxyadenosine triphosphate (dATP), a crowding or stabilizing agent, e.g., polyethylene glycol, e.g., PEG4000, nuclease-free water, a buffer, and a polymerase, e g., a reverse transcriptase. In some embodiments, the template switching reaction mixture comprises a template switching oligonucleotide, dATP, a crowding or stabilizing agent, a buffer, and a reverse transcriptase, e.g., a group II intron reverse transcriptase. In some embodiments, the template switching reaction mixture comprises a template switching oligonucleotide, dATP, a crowding or stabilizing agent, a buffer, and a group II intron reverse transcriptase, e.g., MarathonRT™ or UltraMarathonRT®. In some embodiments, the template switching reaction mixture comprises a template switching oligonucleotide, dATP, PEG4000, a buffer, and MarathonRT™ reverse transcriptase. In some embodiments, the template switching reaction mixture comprises a template switching oligonucleotide, dATP, PEG4000, a buffer, and UltraMarathonRT® reverse transcriptase.

[0104] In some embodiments, the template switching reaction mixture comprises target RNA, e.g., total cellular RNA, e.g., mRNA. In some embodiments, the template switching reaction mixture comprises total cellular RNA. In some embodiments, the template switching reaction mixture comprises mRNA. In some embodiments, the template switching reactionmixture comprises 1 ng - 20 ng of total cellular RNA. In some embodiments, the template switching reaction mixture comprises 0.1 ng - 20 ng of mRNA.

[0105] In some embodiments, the template switching reaction mixture comprises deoxyribonucleotide triphosphates (dNTPs), e.g., deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), or deoxycytidine triphosphate (dCTP). In some embodiments, the template switching reaction mixture comprises dATP. In some embodiments, the template switching reaction mixture comprises dGTP. In some embodiments, the template switching reaction mixture comprises dTTP. In some embodiments, the template switching reaction mixture comprises dCTP. In some embodiments, the template switching reaction mixture comprises a mixture of dATP, dTTP, dGTP, and dCTP. In some embodiments, the template switching reaction mixture comprises a dNTP mixture at a concentration of about 0.1 mM to about 10 mM, e g., about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, or 10 mM. In some embodiments, the template switching reaction mixture comprises a dNTP mixture at a concentration of about 0.1 mM. In some embodiments, the template switching reaction mixture comprises a dNTP mixture at a concentration of about 0.5 mM. In some embodiments, the template switching reaction mixture comprises a dNTP mixture at a concentration of about 1 mM. In some embodiments, the template switching reaction mixture comprises a dNTP mixture at a concentration of about 2 mM. In some embodiments, the template switching reaction mixture comprises a dNTP mixture at a concentration of about 5 mM. In some embodiments, the template switching reaction mixture comprises a dNTP mixture at a concentration of about 10 mM. In some embodiments, the template switching reaction mixture comprises a dNTP mixture at a concentration of 0.5 mM.

[0106] In some embodiments, the template switching reaction mixture comprises an oligonucleotide primer, e.g., a reverse transcription primer. In some embodiments, the template switching reaction mixture comprises a reverse transcription primer. In some embodiments, the template switching reaction mixture comprises a reverse transcription primer at a concentration of about 0.05 pM to about 5 pM, e.g., about 0.05 pM, 0.1 pM, 0.5 pM, 1 pM, 2 pM, or 5 pM. In some embodiments, the template switching reaction mixture comprises a reverse transcription primer at a concentration of about 0.05 pM. In some embodiments, the template switching reaction mixture comprises a reverse transcription primer at a concentration of about 0.1 pM. In some embodiments, the template switching reaction mixture comprises a reverse transcriptionprimer at a concentration of about 0.5 pM. In some embodiments, the template switching reaction mixture comprises a reverse transcription primer at a concentration of about 1 pM. In some embodiments, the template switching reaction mixture comprises a reverse transcription primer at a concentration of about 2 pM. In some embodiments, the template switching reaction mixture comprises a reverse transcription primer at a concentration of about 5 pM. In some embodiments, the template switching reaction mixture comprises a reverse transcription primer at a concentration of 0.25 pM.

[0107] In some embodiments, the template switching reaction mixture comprises carrier RNA. In some embodiments, the template switching reaction mixture comprises carrier RNA at a concentration of about 1 ng / pL to about 100 ng / pL, e.g., about 1 ng / pL, 5 ng / pL, 10 ng / pL, 25 ng / pL, 50 ng / pL, 75 ng / pL, or 100 ng / pL. In some embodiments, the template switching reaction mixture comprises carrier RNA at a concentration of about 1 ng / pL. In some embodiments, the template switching reaction mixture comprises carrier RNA at a concentration of about 5 ng / pL. In some embodiments, the template switching reaction mixture comprises carrier RNA at a concentration of about 10 ng / pL. In some embodiments, the template switching reaction mixture comprises carrier RNA at a concentration of about 25 ng / pL. In some embodiments, the template switching reaction mixture comprises carrier RNA at a concentration of about 50 ng / pL. In some embodiments, the template switching reaction mixture comprises carrier RNA at a concentration of about 75 ng / pL. In some embodiments, the template switching reaction mixture comprises carrier RNA at a concentration of about 100 ng / pL. In some embodiments, the template switching reaction mixture comprises carrier RNA at a concentration of 5 ng / pL.

[0108] In some embodiments, the template switching reaction mixture comprises an RNase inhibitor, e.g., RNaseOUT. In some embodiments, the template switching reaction mixture comprises RNaseOUT. In some embodiments, the template switching reaction mixture comprises RNaseOUT at a concentration of about 1 unit / pL to about 200 units / pL, e.g., about 1 unit / pL, 2 units / pL, 5 units / pL, 10 units / pL, 50 units / pL, 100 units / pL, or 200 units / pL. In some embodiments, the template switching reaction mixture comprises RNaseOUT at a concentration of about 1 unit / pL. In some embodiments, the template switching reaction mixture comprises RNaseOUT at a concentration of about 2 units / pL. In some embodiments, the template switching reaction mixture comprises RNaseOUT at a concentration of about 5 units / pL. In some embodiments, the template switching reaction mixture comprises RNaseOUT at a concentrationof about 10 units / pL. In some embodiments, the template switching reaction mixture comprises RNaseOUT at a concentration of about 50 units / pL. In some embodiments, the template switching reaction mixture comprises RNaseOUT at a concentration of about 100 units / pL. In some embodiments, the template switching reaction mixture comprises RNaseOUT at a concentration of about 200 units / pL. In some embodiments, the template switching reaction mixture comprises RNaseOUT at a concentration of 20 units / pL.

[0109] In some embodiments, the template switching reaction mixture comprises a complementary DNA (cDNA), e.g. the cDNA product generated from the reverse transcription reaction, e.g., the cDNA product generated from the first reaction step. In some embodiments, the template switching reaction mixture comprises a cDNA that is complementary to the target RNA in the reverse transcription reaction mixture. In some embodiments, the template switching reaction mixture comprises cDNA at a concentration of at least 0.0005 pg / pL, e g., at least 0.0005 pg / pL, 0.005 pg / pL, 0.05 pg / pL, 0.5 pg / pL, or more.

[0110] In some embodiments, the template switching reaction mixture comprises a template switching oligonucleotide (TSO). In some embodiments, the template switching reaction mixture comprises a TSO at a concentration of about 0.1 pM to about 10 pM, e.g., about 0.1 pM, 0.5 pM, 1 pM, 2 pM, 5 pM, or 10 pM. In some embodiments, the template switching reaction mixture comprises a TSO at a concentration of about 0.1 pM. In some embodiments, the template switching reaction mixture comprises a TSO at a concentration of about 0.5 pM. In some embodiments, the template switching reaction mixture comprises a TSO at a concentration of about 1 pM. In some embodiments, the template switching reaction mixture comprises a TSO at a concentration of about 2 pM. In some embodiments, the template switching reaction mixture comprises a TSO at a concentration of about 5 pM. In some embodiments, the template switching reaction mixture comprises a TSO at a concentration of about 10 pM. In some embodiments, the template switching reaction mixture comprises a TSO at a concentration of 1 pM.

[0111] In some embodiments, the template switching reaction mixture comprises additional deoxyadenosine triphosphate (dATP). In some embodiments, the template switching reaction mixture comprises additional dATP at a concentration of about 0.1 mM to about 10 mM, e.g., about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, or 10 mM. In some embodiments, the template switching reaction mixture comprises additional dATP at a concentration of about 0.1mM. In some embodiments, the template switching reaction mixture comprises additional dATP at a concentration of about 0.5 mM. In some embodiments, the template switching reaction mixture comprises additional dATP at a concentration of about 1 mM. In some embodiments, the template switching reaction mixture comprises additional dATP at a concentration of about 2 mM. In some embodiments, the template switching reaction mixture comprises additional dATP at a concentration of about 5 mM. In some embodiments, the template switching reaction mixture comprises additional dATP at a concentration of about 10 mM. In some embodiments, the template switching reaction mixture comprises additional dATP at a concentration of 1 mM. In some embodiments, the amount of dATP present in the template switching reaction mixture is greater than the amount of dCTP, dTTP, or dGTP For example, dATP may be present in the template switching reaction mixture at a concentration that is two times, three times, four times, or more greater than the concentration of dCTP, dTTP, or dGTP. In some embodiments, the amount of dATP present in the template switching reaction mixture is greater than the amount of dCTP. In some embodiments, the amount of dATP present in the template switching reaction mixture is greater than the amount of dTTP. In some embodiments, the amount of dATP present in the template switching reaction mixture is greater than the amount of dGTP. In some embodiments, the amount of dATP present in the template switching reaction mixture is two times greater than the amount of dCTP In some embodiments, the amount of dATP present in the template switching reaction mixture is two times greater than the amount of dTTP. In some embodiments, the amount of dATP present in the template switching reaction mixture is two times greater than the amount of dGTP In some embodiments, the amount of dATP present in the template switching reaction mixture is three times greater than the amount of dCTP. In some embodiments, the amount of dATP present in the template switching reaction mixture is three times greater than the amount of dTTP. In some embodiments, the amount of dATP present in the template switching reaction mixture is three times greater than the amount of dGTP. In some embodiments, the amount of dATP present in the template switching reaction mixture is four times greater than the amount of dCTP In some embodiments, the amount of dATP present in the template switching reaction mixture is four times greater than the amount of dTTP. In some embodiments, the amount of dATP present in the template switching reaction mixture is four times greater than the amount of dGTP In some embodiments, the amount of dATP present in the template switching reaction mixture is more than four times greater than the amount of dCTP.In some embodiments, the amount of dATP present in the template switching reaction mixture is more than four times greater than the amount of dTTP. In some embodiments, the amount of dATP present in the template switching reaction mixture is more than four times greater than the amount of dGTP In some embodiments, the concentration of dATP present in the template switching reaction mixture is greater than the concentration of dCTP. In some embodiments, the concentration of dATP present in the template switching reaction mixture is greater than the concentration of dTTP In some embodiments, the concentration of dATP present in the template switching reaction mixture is greater than the concentration of dGTP. In some embodiments, the concentration of dATP present in the template switching reaction mixture is two times greater than the concentration of dCTP. In some embodiments, the concentration of dATP present in the template switching reaction mixture is two times greater than the concentration of dTTP In some embodiments, the concentration of dATP present in the template switching reaction mixture is two times greater than the concentration of dGTP In some embodiments, the concentration of dATP present in the template switching reaction mixture is three times greater than the concentration of dCTP. In some embodiments, the concentration of dATP present in the template switching reaction mixture is three times greater than the concentration of dTTP. In some embodiments, the concentration of dATP present in the template switching reaction mixture is three times greater than the concentration of dGTP. In some embodiments, the concentration of dATP present in the template switching reaction mixture is four times greater than the concentration of dCTP. In some embodiments, the concentration of dATP present in the template switching reaction mixture is four times greater than the concentration of dTTP. In some embodiments, the concentration of dATP present in the template switching reaction mixture is four times greater than the concentration of dGTP. In some embodiments, the concentration of dATP present in the template switching reaction mixture is more than four times greater than the concentration of dCTP. In some embodiments, the concentration of dATP present in the template switching reaction mixture is more than four times greater than the concentration of dTTP In some embodiments, the concentration of dATP present in the template switching reaction mixture is more than four times greater than the concentration of dGTP.

[0112] In some embodiments, the template switching reaction mixture comprises a crowding or stabilizing agent, e.g., polyethylene glycol, e.g., PEG4000. In some embodiments, the template switching reaction mixture comprises polyethylene glycol. In some embodiments,the template switching reaction mixture comprises PEG4000. In some embodiments, the template switching reaction mixture comprises PEG4000 at a concentration of about 1% (vol / vol) to about 50% (vol / vol), e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the template switching reaction mixture comprises PEG4000 at a concentration of about 1%. In some embodiments, the template switching reaction mixture comprises PEG4000 at a concentration of about 5%. In some embodiments, the template switching reaction mixture comprises PEG4000 at a concentration of about 10%. In some embodiments, the template switching reaction mixture comprises PEG4000 at a concentration of about 15%. In some embodiments, the template switching reaction mixture comprises PEG4000 at a concentration of about 20%. In some embodiments, the template switching reaction mixture comprises PEG4000 at a concentration of about 25%. In some embodiments, the template switching reaction mixture comprises PEG4000 at a concentration of about 30%. In some embodiments, the template switching reaction mixture comprises PEG4000 at a concentration of about 35%. In some embodiments, the template switching reaction mixture comprises PEG4000 at a concentration of about 40%. In some embodiments, the template switching reaction mixture comprises PEG4000 at a concentration of about 45%. In some embodiments, the template switching reaction mixture comprises PEG4000 at a concentration of about 50%. In some embodiments, the template switching reaction mixture comprises PEG4000 at a concentration of 1% to 50% (vol / vol), e.g., 10% (vol / vol).

[0113] In some embodiments, the template switching reaction mixture comprises a buffer. In some embodiments, the template switching reaction mixture comprises a buffering agent, e.g., Tris-HCl. In some embodiments, the template switching reaction mixture comprises Tris-HCl. In some embodiments, the template switching reaction mixture comprises Tris-HCl at a concentration of about 5 mM to about 500 mM, e g., about 5 mM, 10 mM, 50 mM, 100 mM, 250 mM, or 500 mM. In some embodiments, the template switching reaction mixture comprises Tris-HCl at a concentration of about 5 mM. In some embodiments, the template switching reaction mixture comprises Tris-HCl at a concentration of about 10 mM. In some embodiments, the template switching reaction mixture comprises Tris-HCl at a concentration of about 50 mM. In some embodiments, the template switching reaction mixture comprises Tris-HCl at a concentration of about 100 mM. In some embodiments, the template switching reaction mixture comprises Tris-HCl at a concentration of about 250 mM. In some embodiments, the templateswitching reaction mixture comprises Tris-HCl at a concentration of about 500 mM. In some embodiments, the template switching reaction mixture comprises a salt solution, e.g., KC1, LiCl, or MgCh. In some embodiments, the template switching reaction mixture comprises KC1. In some embodiments, the template switching reaction mixture comprises KC1 at a concentration of about 20 mM to about IM, e.g., about 20 mM, 50 mM, 100 mM, 200 mM, 500 mM, or IM. In some embodiments, the template switching reaction mixture comprises KC1 at a concentration of about 20 mM. In some embodiments, the template switching reaction mixture comprises KC1 at a concentration of about 50 mM. In some embodiments, the template switching reaction mixture comprises KC1 at a concentration of about 100 mM. In some embodiments, the template switching reaction mixture comprises KC1 at a concentration of about 200 mM. In some embodiments, the template switching reaction mixture comprises KC1 at a concentration of about 500 mM. In some embodiments, the template switching reaction mixture comprises KC1 at a concentration of about IM. In some embodiments, the template switching reaction mixture comprises LiCl. In some embodiments, the template switching reaction mixture comprises LiCl at a concentration of about 20 mM to about IM, e.g., about 20 mM, 50 mM, 100 mM, 200 mM, 500 mM, or IM. In some embodiments, the template switching reaction mixture comprises LiCl at a concentration of about 20 mM. In some embodiments, the template switching reaction mixture comprises LiCl at a concentration of about 50 mM. In some embodiments, the template switching reaction mixture comprises LiCl at a concentration of about 100 mM. In some embodiments, the template switching reaction mixture comprises LiCl at a concentration of about 200 mM. In some embodiments, the template switching reaction mixture comprises LiCl at a concentration of about 500 mM. In some embodiments, the template switching reaction mixture comprises LiCl at a concentration of about IM. In some embodiments, the template switching reaction mixture comprises MgCh. In some embodiments, the template switching reaction mixture comprises MgCh at a concentration of about 0. 1 mM to about 50 mM, e.g., about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 25 mM, or 50 mM. In some embodiments, the template switching reaction mixture comprises MgCh at a concentration of about 0.1 mM. In some embodiments, the template switching reaction mixture comprises MgCh at a concentration of about 0.5 mM. In some embodiments, the template switching reaction mixture comprises MgCh at a concentration of about 1 mM. In some embodiments, the template switching reaction mixture comprises MgCh at a concentration of about 5 mM. In some embodiments, the templateswitching reaction mixture comprises MgCh at a concentration of about 10 mM. In some embodiments, the template switching reaction mixture comprises MgCh at a concentration of about 25 mM. In some embodiments, the template switching reaction mixture comprises MgCh at a concentration of about 50 mM. In some embodiments, the template switching reaction mixture comprises a reducing agent, e.g., dithiothreitol (DTT). In some embodiments, the template switching reaction mixture comprises DTT. In some embodiments, the template switching reaction mixture comprises DTT at a concentration of about 0.5 mM to about 50 mM, e.g., about 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 25 mM, or 50 mM. In some embodiments, the template switching reaction mixture comprises DTT at a concentration of about 0.5 mM. In some embodiments, the template switching reaction mixture comprises DTT at a concentration of about 1 mM. In some embodiments, the template switching reaction mixture comprises DTT at a concentration of about 2 mM. In some embodiments, the template switching reaction mixture comprises DTT at a concentration of about 5 mM. In some embodiments, the template switching reaction mixture comprises DTT at a concentration of about 10 mM. In some embodiments, the template switching reaction mixture comprises DTT at a concentration of about 25 mM. In some embodiments, the template switching reaction mixture comprises DTT at a concentration of about 50 mM. In some embodiments, the template switching reaction mixture comprises a crowding or stabilizing reagent, e.g. glycerol. In some embodiments, the template switching reaction mixture comprises glycerol. In some embodiments, the template switching reaction mixture comprises glycerol at a concentration of about 1% (vol / vol) to about 50% (vol / vol), e.g., about 1%, 5%, 10%, 25%, or 50%. In some embodiments, the template switching reaction mixture comprises glycerol at a concentration of about 1%. In some embodiments, the template switching reaction mixture comprises glycerol at a concentration of about 5%. In some embodiments, the template switching reaction mixture comprises glycerol at a concentration of about 10%. In some embodiments, the template switching reaction mixture comprises glycerol at a concentration of about 25%. In some embodiments, the template switching reaction mixture comprises glycerol at a concentration of about 50%. In some embodiments, the template switching reaction buffer comprises 50 mM Tris-HCl pH 8.3, 100 mM KC1, 4 mM of MgCh, 5 mM of DTT, 10% glycerol, and 10% PEG4000. In some embodiments, the ionic strength of the template switching reaction mixture is less than the ionic strength of the reverse transcription reaction mixture, e.g., the ionic strength of the template switching reaction mixture is two times,three times, four times, or more lower than the ionic strength of the reverse transcription reaction mixture. In some embodiments, the ionic strength of the template switching reaction mixture is two times lower than the ionic strength of the reverse transcription reaction mixture. In some embodiments, the ionic strength of the template switching reaction mixture is three times lower than the ionic strength of the reverse transcription reaction mixture. In some embodiments, the ionic strength of the template switching reaction mixture is four times lower than the ionic strength of the reverse transcription reaction mixture. In some embodiments, the ionic strength of the template switching reaction mixture is more than four times lower than the ionic strength of the reverse transcription reaction mixture.

[0114] In some embodiments, the template switching reaction mixture comprises a polymerase, e.g., a DNA polymerase or a reverse transcriptase. In some embodiments, the template switching reaction mixture comprises a DNA polymerase. In some embodiments, the template switching reaction mixture comprises a reverse transcriptase, e.g., an MMLV reverse transcriptase or a group II intron reverse transcriptase. In some embodiments, the reverse transcriptase is an MMLV reverse transcriptase. In some embodiments, the template switching reaction mixture comprises SuperScript™, e.g., SuperScript™, SuperScript™ II, SuperScript™ III, or SuperScript™ IV. In some embodiments, the template switching reaction mixture comprises a group II intron reverse transcriptase, e.g., MarathonRT™ or UltraMarathonRT®. In some embodiments, the template switching reaction mixture comprises MarathonRT™ reverse transcriptase. In some embodiments, the template switching reaction mixture comprises UltraMarathonRT® reverse transcriptase. In some embodiments, the template switching reaction mixture comprises Induro® RT. In some embodiments, the template switching reaction mixture comprises about 0.1 units to about 100 units of a polymerase, e.g., reverse transcriptase, e.g., about 0.1, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or more units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 0.1-1,000 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 1-900 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 10-800 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 100-700 units of reverse transcriptase. In some embodiments, the template switchingreaction mixture comprises between about 200-600 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 300-500 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 500-1,000 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 1-500 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 10-200 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 100-200 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 10-60 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 20-50 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 30-40 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 40-50 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises between about 30-60 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 0.1 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 1 unit of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 2 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 5 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 10 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 20 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 30 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 40 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 50 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 60 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 70 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 80 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 90 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about100 units of reverse transcriptase. In some embodiments, the template switching reaction mixture comprises about 40 units of MarathonRT™. In some embodiments, the template switching reaction mixture comprises about 40 units of UltraMarathonRT®. In some embodiments, the template switching reaction mixture comprises a reverse transcriptase at a concentration of about 0.1 units / pL to about 20 units / pL, e.g., about 0.1 units / pL, 0.5 units / pL, 1 unit / pL, 2 units / pL, 5 units / pL, 10 units / pL, or 20 units / pL. In some embodiments, the template switching reaction mixture comprises a reverse transcriptase at a concentration of about 0.1 units / pL. In some embodiments, the template switching reaction mixture comprises a reverse transcriptase at a concentration of about 0.5 units / pL. In some embodiments, the template switching reaction mixture comprises a reverse transcriptase at a concentration of about 1 unit / pL. In some embodiments, the template switching reaction mixture comprises a reverse transcriptase at a concentration of about 2 units / pL. In some embodiments, the template switching reaction mixture comprises a reverse transcriptase at a concentration of about 5 units / pL. In some embodiments, the template switching reaction mixture comprises a reverse transcriptase at a concentration of about 10 units / pL. In some embodiments, the template switching reaction mixture comprises a reverse transcriptase at a concentration of about 20 units / pL. In some embodiments, the template switching reaction mixture comprises UltraMarathonRT® reverse transcriptase at a concentration of 2 units / pL. In some embodiments, the template switching reaction mixture comprises the same polymerase as present in the reverse transcription reaction mixture. In some embodiments, the template switching reaction mixture comprises a different polymerase than present in the reverse transcription reaction mixture.First reaction steps

[0115] In one aspect, the method for two-step reverse transcription and template switching described herein includes a first reaction step, e.g., a reverse transcription reaction step. In some embodiments, the first reaction step comprises a reverse transcription step. In some embodiments, the reverse transcription step comprises one or more of the following: (i) annealing of an oligonucleotide primer to a target polynucleotide; (ii) preparing a reverse transcription reaction mixture; and (iii) incubating the reverse transcription reaction mixture to perform reverse transcription. In some embodiments, the reverse transcription step comprises one of (i)-(iii). In some embodiments, the reverse transcription step comprises two of (i)-(iii). Insome embodiments, the reverse transcription step comprises each of (i)-(iii). In some embodiments, the reverse transcription step comprises (i). In some embodiments, the reverse transcription step comprises (ii). In some embodiments, the reverse transcription step comprises (iii).

[0116] In some embodiments, the reverse transcription step comprises annealing, e.g., hybridizing or base pairing, of an oligonucleotide primer to a target polynucleotide. In some embodiments, the oligonucleotide primer is a reverse transcription primer. In some embodiments, the target polynucleotide is a plurality of target polynucleotides. In some embodiments, the target polynucleotide is DNA. In some embodiments, the DNAis total cellular DNA. In some embodiments, the target polynucleotide is RNA. In some embodiments, the RNA is total cellular RNA. In some embodiments, the RNA is mRNA. In some embodiments, annealing of an oligonucleotide primer to a target polynucleotide comprises generating a mixture comprising an oligonucleotide primer, e.g., a reverse transcription primer, and a target polynucleotide, e.g., a plurality of target polynucleotides. In some embodiments, the mixture comprises: an oligonucleotide primer, e.g. a reverse transcription primer; a target polynucleotide, e.g., a plurality of target polynucleotides; a deoxyribonucleotide triphosphate (dNTP) solution; and purified nuclease-free water. In some embodiments, the oligonucleotide primer, target polynucleotide, dNTP solution, or nuclease-free water is thawed prior to being added to the mixture. In some embodiments, the oligonucleotide primer, target polynucleotide, dNTP solution, or nuclease-free water is mixed prior to being added to the mixture. In some embodiments, the oligonucleotide primer, target polynucleotide, dNTP solution, or nuclease-free water is centrifuged prior to being added to the mixture. In some embodiments, the mixture is generated by adding the oligonucleotide primer, target polynucleotide, dNTP solution, and nuclease-free water to a tube, e g., a 0.2 mL, 0.5 mL, 1 mb, 1.5 mL, 2 mL, 5 m , 15 mb, or 50 mb tube, e.g., a microcentrifuge tube or a polymerase chain reaction (PCR) tube. In some embodiments, the mixture is mixed by gently tapping the tube after adding the components. In some embodiments, the mixture is centrifuged after adding the components. In some embodiments, the mixture is incubated at about 95°C, e.g., about 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, or 100°C. In some embodiments, the mixture is incubated for about 30 seconds, e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,'ll38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, or more seconds. In some embodiments, the mixture is snap cooled on ice following incubation.

[0117] In some embodiments, the reverse transcription step comprises preparing a reverse transcription mixture, e.g., a reverse transcription reaction mixture described herein. In some embodiments, preparing the reverse transcription reaction mixture comprises adding to the annealed oligonucleotide primer and target RNA mixture: carrier RNA; a buffer; a polymerase, e.g., a DNA polymerase, e.g., a reverse transcriptase; an RNase inhibitor; and purified nuclease- free water. In some embodiments, the carrier RNA, buffer, polymerase, RNAse inhibitor, or nuclease-free water is thawed prior to being added to the mixture. In some embodiments, the carrier RNA, buffer, polymerase, RNAse inhibitor, or nuclease-free water is mixed prior to being added to the mixture. In some embodiments, the carrier RNA, buffer, polymerase, RNAse inhibitor, or nuclease-free water is centrifuged prior to being added to the mixture. In some embodiments, the carrier RNA, buffer, polymerase, RNAse inhibitor, and nuclease-free water is added to the annealed oligonucleotide primer and target RNA mixture to generate a reverse transcription reaction mixture. In some embodiments, the reverse transcription reaction mixture is mixed following addition of the carrier RNA, buffer, polymerase, RNAse inhibitor, and nuclease-free water to the annealed oligonucleotide primer and target RNA mixture. In some embodiments, the reverse transcription reaction mixture is centrifuged following addition of the carrier RNA, buffer, polymerase, RNAse inhibitor, and nuclease-free water to the annealed oligonucleotide primer and target RNA mixture.

[0118] In some embodiments, the reverse transcription step comprises incubating the reverse transcription reaction mixture to perform reverse transcription. In some embodiments, the reverse transcription reaction is carried out at a temperature that allows the polymerase to reverse transcribe the target RNA, e.g., a temperature that permits reverse transcriptase activity of the polymerase. For example, the reverse transcription reaction may be carried out at a temperature of about 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C ,52°C, 53°C, 54°C, 55°C, or greater than 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C or greater. In some embodiments, the reverse transcription reaction is carried out at atemperature of less than 4°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 5°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 6°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 7°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 8°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 9°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 10°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 11°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 12°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 13°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 14°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 15°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 16°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 17°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 18°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 19°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 20°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 21°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 22°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 23°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 24°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 25°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 26°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 27°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 28°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 29°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 30°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 31°C. In some embodiments, the reverse transcription reaction is carried out at atemperature of about 32°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 33°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 34°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 35°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 36°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 37°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 38°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 39°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 40°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 41 °C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 42°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 43°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 44°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 45°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 46°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 47°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 48°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 49°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 50°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 51 °C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 52°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 53°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 54°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 55°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 56°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 57°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 58°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 59°C. In some embodiments, the reverse transcription reaction is carriedout at a temperature of about 60°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 61 °C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 62°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 63 °C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 64°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 65°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 66°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 67°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 68°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 69°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 70°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 71°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 72°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 73°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 74°C. In some embodiments, the reverse transcription reaction is carried out at a temperature of about 75°C. In some embodiments, the reverse transcription reaction is carried out at a temperature greater than 75°C. In some embodiments, the reverse transcription reaction is incubated for an amount of time sufficient for cDNAto by synthesized before being contacted with the template switching reaction components, e.g., incubated for at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, or 150 minutes or more. In some embodiments, the reverse transcription reaction is incubated for at least 10 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 20 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 30 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 40 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 50 minutes before being contacted with thetemplate switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 60 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 70 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 80 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 90 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 100 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 110 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 120 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 130 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 140 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for at least 150 minutes before being contacted with the template switching reaction components. In some embodiments, the reverse transcription reaction is incubated for greater than 150 minutes before being contacted with the template switching reaction components. In some embodiments, the polymerase is not inactivated, e.g., inactivated by heat or a chemical, following reverse transcription.

[0119] In some embodiments, the first reaction mixture is separated or purified after incubating for a period of time, e g., before being contacted with a template switching reaction mixture. In some embodiments, the purification comprises filtration, centrifugation, chromatography, or electrophoresis. In some embodiments, the purification comprises filtration. In some embodiments, the purification comprises centrifugation. In some embodiments, the purification comprises chromatography. In some embodiments, the purification comprises electrophoresis. In some embodiments, the purification comprises separating cDNA, e.g., a plurality of cDNA molecules, from a reverse transcription reaction mixture. In someembodiments, the purification comprises separating a cDNA product, e.g., a plurality of cDNA products, from a reverse transcription reaction mixture.Second reaction steps

[0120] In one aspect, the method for two-step reverse transcription and template switching described herein includes a second reaction step, e.g., a template switching reaction step. In some embodiments, the second reaction step comprises a template switching step. In some embodiments, the template switching step comprises one or more of the following: (i) preparing a template switching reaction mixture; and (ii) incubating the template switching reaction mixture to perform template switching. In some embodiments, the template switching step comprises one of (i)-(ii). In some embodiments, the reverse transcription step comprises two of (i)-(ii). In some embodiments, the reverse transcription step comprises each of (i)-(ii). In some embodiments, the reverse transcription step comprises (i). In some embodiments, the reverse transcription step comprises (ii). In some embodiments, the template switching step comprises providing a template switching oligonucleotide after a reverse transcription reaction is carried out for a period of time, e g., about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more minutes after initiation of a reverse transcription reaction.

[0121] In some embodiments, the template switching reaction step comprises preparing a template switching reaction mixture, e g., a template switching reaction mixture described herein. In some embodiments, preparing the template switching reaction mixture comprises adding to the reverse transcription reaction mixture: additional polymerase, e.g., an additional amount of the same polymerase used for reverse transcription, e.g., a different polymerase than the polymerase used for reverse transcription; additional deoxyadenosine triphosphate (dATP); a template switching oligonucleotide (TSO); a crowding or stabilizing agent, e g., polyethylene glycol; and a buffer. In some embodiments, the polymerase, dATP, TSO, crowding agent, or buffer is thawed prior to being added to the reverse transcription reaction mixture. In some embodiments, the polymerase, dATP, TSO, crowding agent, or buffer is mixed prior to being added to the reverse transcription reaction mixture. In some embodiments, the polymerase, dATP, TSO, crowding agent, or buffer is centrifuged prior to being added to the reverse transcription reaction mixture. In some embodiments, the polymerase, dATP, TSO, crowding agent, or buffer is added to the reverse transcription reaction mixture to generate a template switching reactionmixture. In some embodiments, the template switching reaction mixture is mixed following addition of the polymerase, dATP, TSO, crowding agent or buffer. In some embodiments, the template switching reaction mixture is centrifuged following addition of the polymerase, dATP, TSO, crowding agent or buffer.

[0122] In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture, e.g., after a period of time, e.g., about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more minutes after initiation of a reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 1-100 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 10-90 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 1-60 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 20-50 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 30-50 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 1 minute after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 2 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 5 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 10 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 20 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 30 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 40 minutes after initiation of the reverse transcription reaction. In someembodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 50 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 60 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 70 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 80 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture about 90 minutes after initiation of the reverse transcription reaction. In some embodiments, the template switching reaction mixture is added to the reverse transcription reaction mixture more than 90 minutes after initiation of the reverse transcription reaction.

[0123] In some embodiments, a template switching oligonucleotide (TSO) is added to the reverse transcription reaction mixture, e.g., after a period of time, e.g., about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more minutes after initiation of a reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 1-100 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 10-90 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 1-60 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 20-50 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 30-50 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 1 minute after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 2 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 5 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 10 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 20 minutes after initiation of thereverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 30 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 40 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 50 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 60 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 70 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 80 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 90 minutes after initiation of the reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture more than 90 minutes after initiation of the reverse transcription reaction.

[0124] In some embodiments, a polymerase enzyme, e.g., reverse transcriptase, e.g., MarathonRT™ or UltraMarathonRT®, is added to the reverse transcription reaction mixture, e.g., after a period of time, e.g., about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more minutes after initiation of a reverse transcription reaction. For example, about 1 to about 1,000 units of reverse transcriptase, e.g., about 1, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or more units of reverse transcriptase, may be added to the reverse transcription reaction mixture a period of time after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 1-100 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 10-90 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 1-60 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 20-50 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 30-50 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcriptionreaction mixture about 1 minute after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 2 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 5 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 10 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 20 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 30 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 40 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 50 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 60 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 70 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 80 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture about 90 minutes after initiation of the reverse transcription reaction. In some embodiments, a reverse transcriptase is added to the reverse transcription reaction mixture more than 90 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 1-100 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 10-90 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 1-60 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 20-50 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 30-50 minutes after initiation of the reversetranscription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 1 minute after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 2 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 5 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 10 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 20 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 30 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 40 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 50 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 60 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 70 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 80 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture about 90 minutes after initiation of the reverse transcription reaction. In some embodiments, MarathonRT™ is added to the reverse transcription reaction mixture more than 90 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 1-100 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 10-90 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 1-60 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 20-50 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reactionmixture about 30-50 minutes after initiation of the reverse transcription reaction. Tn some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 1 minute after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 2 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 5 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 10 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 20 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 30 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 40 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 50 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 60 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 70 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 80 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture about 90 minutes after initiation of the reverse transcription reaction. In some embodiments, UltraMarathonRT® is added to the reverse transcription reaction mixture more than 90 minutes after initiation of the reverse transcription reaction.

[0125] In some embodiments, a deoxynucleoside triphosphate, e.g., deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), or deoxy guanosine triphosphate (dGTP), is added to the reverse transcription reaction mixture, e.g., after a period of time, e.g., about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more minutes after initiation of a reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 1-100 minutes afterinitiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 10-90 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 1-60 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 20-50 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 30-50 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 1 minute after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 2 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 5 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 10 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 20 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 30 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 40 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 50 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 60 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 70 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture about 80 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reversetranscription reaction mixture about 90 minutes after initiation of the reverse transcription reaction. In some embodiments, a deoxynucleoside triphosphate is added to the reverse transcription reaction mixture more than 90 minutes after initiation of the reverse transcription reaction. In some embodiments, deoxycytidine triphosphate (dCTP) is added to the reverse transcription reaction mixture about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more minutes after initiation of a reverse transcription reaction. In some embodiments, deoxythymidine triphosphate (dTTP) is added to the reverse transcription reaction mixture about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more minutes after initiation of a reverse transcription reaction. In some embodiments, deoxyguanosine triphosphate (dGTP) is added to the reverse transcription reaction mixture about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more minutes after initiation of a reverse transcription reaction. In some embodiments, deoxyadenosine triphosphate (dATP) is added to the reverse transcription reaction mixture about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more minutes after initiation of a reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 1-100 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 10-90 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 1-60 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 20-50 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 30-50 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 1 minute after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 2 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 5 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 10 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 20 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 30 minutes after initiation of the reverse transcription reaction. In someembodiments, dATP is added to the reverse transcription reaction mixture about 40 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 50 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 60 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 70 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 80 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture about 90 minutes after initiation of the reverse transcription reaction. In some embodiments, dATP is added to the reverse transcription reaction mixture more than 90 minutes after initiation of the reverse transcription reaction.

[0126] In some embodiments, a crowding agent, e.g., polyethylene glycol, e.g., PEG4000, is added to the reverse transcription reaction mixture, e.g., after a period of time, e.g., about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more minutes after initiation of a reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more minutes after initiation of a reverse transcription reaction.

[0127] In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 1-100 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 10-90 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 1-60 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 20-50 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 30-50 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 1 minute after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 2 minutes after initiation of the reverse transcription reaction. In someembodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 5 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 10 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 20 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 30 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 40 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 50 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 60 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 70 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 80 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture about 90 minutes after initiation of the reverse transcription reaction. In some embodiments, a polyethylene glycol is added to the reverse transcription reaction mixture more than 90 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more minutes after initiation of a reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 1-100 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 10-90 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 1-60 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 20-50 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 30-50 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to thereverse transcription reaction mixture about 1 minute after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 2 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 5 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 10 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 20 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 30 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 40 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 50 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 60 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 70 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 80 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture about 90 minutes after initiation of the reverse transcription reaction. In some embodiments, PEG4000 is added to the reverse transcription reaction mixture more than 90 minutes after initiation of the reverse transcription reaction.

[0128] In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture, e.g., after a period of time, e.g., about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more minutes after initiation of a reverse transcription reaction. In some embodiments, a TSO is added to the reverse transcription reaction mixture about 1-100 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 10-90 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcriptionreaction mixture about 1-60 minutes after initiation of the reverse transcription reaction. Tn some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 20-50 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 30-50 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 1 minute after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 2 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 5 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 10 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 20 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 30 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 40 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 50 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 60 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 70 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reversetranscription reaction mixture about 80 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture about 90 minutes after initiation of the reverse transcription reaction. In some embodiments, a buffer with lower ionic strength than the reverse transcription reaction buffer is added to the reverse transcription reaction mixture more than 90 minutes after initiation of the reverse transcription reaction.

[0129] In some embodiments, the template switching reaction mixture is incubated at a temperature that allows the polymerase to perform non-templated nucleotide addition to the cDNA and switch templates, e.g., a temperature that permits template switching activity of the polymerase. For example, the template switching reaction mixture may be incubated at a temperature of about 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C,17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C,32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C,47°C, 48°C, 49°C, 50°C, 51°C ,52°C, 53°C, 54°C, 55°C, or greater than 55°C, 56°C, 57°C, 58°C,59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C,74°C, 75°C or greater. In some embodiments, the template switching reaction mixture is incubated at a temperature of less than 4°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 5°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 6°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 7°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 8°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 9°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 10°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 11°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 12°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 13°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 14°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 15°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 16°C. In some embodiments, the template switching reaction mixture isincubated at a temperature of about 17°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 18°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 19°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 20°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 21 °C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 22°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 23 °C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 24°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 25°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 26°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 27°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 28°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 29°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 30°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 31°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 32°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 33°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 34°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 35°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 36°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 37°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 38°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 39°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 40°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 41 °C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 42°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 43°C. In some embodiments,the template switching reaction mixture is incubated at a temperature of about 44°C. Tn some embodiments, the template switching reaction mixture is incubated at a temperature of about 45°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 46°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 47°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 48°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 49°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 50°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 51 °C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 52°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 53°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 54°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 55°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 56°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 57°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 58°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 59°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 60°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 61°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 62°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 63°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 64°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 65°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 66°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 67°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 68°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 69°C. In some embodiments, the template switching reaction mixture is incubated at atemperature of about 70°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 71 °C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 72°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 73°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 74°C. In some embodiments, the template switching reaction mixture is incubated at a temperature of about 75°C. In some embodiments, the template switching reaction mixture is incubated at a temperature greater than 75 °C.

[0130] In some embodiments, the template switching reaction step comprises incubating the template switching reaction mixture to perform template switching. In some embodiments, the template switching reaction is carried out at a temperature that allows the polymerase to perform non-templated nucleotide addition to the cDNA and switch templates, e.g., a temperature that permits template switching activity of the polymerase. For example, the template switching reaction may be carried out at a temperature of about 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C,38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C ,52°C,53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C,68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C or greater. In some embodiments, the template switching reaction is carried out at a temperature of less than 4°C. In some embodiments, the template switching reaction is carried out at a temperature of about 4°C. In some embodiments, the template switching reaction is carried out at a temperature of about 5°C. In some embodiments, the template switching reaction is carried out at a temperature of about 6°C. In some embodiments, the template switching reaction is carried out at a temperature of about 7°C. In some embodiments, the template switching reaction is carried out at a temperature of about 8°C. In some embodiments, the template switching reaction is carried out at a temperature of about 9°C. In some embodiments, the template switching reaction is carried out at a temperature of about 10°C. In some embodiments, the template switching reaction is carried out at a temperature of about 11 °C. In some embodiments, the template switching reaction is carried out at a temperature of about 12°C. In some embodiments, the template switching reaction is carried out at a temperature of about 13°C. In some embodiments, the template switching reaction iscarried out at a temperature of about 14°C. In some embodiments, the template switching reaction is carried out at a temperature of about 15°C. In some embodiments, the template switching reaction is carried out at a temperature of about 16°C. In some embodiments, the template switching reaction is carried out at a temperature of about 17°C. In some embodiments, the template switching reaction is carried out at a temperature of about 18°C. In some embodiments, the template switching reaction is carried out at a temperature of about 19°C. In some embodiments, the template switching reaction is carried out at a temperature of about 20°C. In some embodiments, the template switching reaction is carried out at a temperature of about 21 °C. In some embodiments, the template switching reaction is carried out at a temperature of about 22°C. In some embodiments, the template switching reaction is carried out at a temperature of about 23 °C. In some embodiments, the template switching reaction is carried out at a temperature of about 24°C. In some embodiments, the template switching reaction is carried out at a temperature of about 25°C. In some embodiments, the template switching reaction is carried out at a temperature of about 26°C. In some embodiments, the template switching reaction is carried out at a temperature of about 27°C. In some embodiments, the template switching reaction is carried out at a temperature of about 28°C. In some embodiments, the template switching reaction is carried out at a temperature of about 29°C. In some embodiments, the template switching reaction is carried out at a temperature of about 30°C. In some embodiments, the template switching reaction is carried out at a temperature of about 31°C. In some embodiments, the template switching reaction is carried out at a temperature of about 32°C. In some embodiments, the template switching reaction is carried out at a temperature of about 33°C. In some embodiments, the template switching reaction is carried out at a temperature of about 34°C. In some embodiments, the template switching reaction is carried out at a temperature of about 35°C. In some embodiments, the template switching reaction is carried out at a temperature of about 36°C. In some embodiments, the template switching reaction is carried out at a temperature of about 37°C. In some embodiments, the template switching reaction is carried out at a temperature of about 38°C. In some embodiments, the template switching reaction is carried out at a temperature of about 39°C. In some embodiments, the template switching reaction is carried out at a temperature of about 40°C. In some embodiments, the template switching reaction is carried out at a temperature of about 41 °C. In some embodiments, the template switching reaction is carried out at a temperature of about42°C. In some embodiments, the template switching reaction is carried out at a temperature of about 43°C. In some embodiments, the template switching reaction is carried out at a temperature of about 44°C. In some embodiments, the template switching reaction is carried out at a temperature of about 45°C. In some embodiments, the template switching reaction is carried out at a temperature of about 46°C. In some embodiments, the template switching reaction is carried out at a temperature of about 47°C. In some embodiments, the template switching reaction is carried out at a temperature of about 48°C. In some embodiments, the template switching reaction is carried out at a temperature of about 49°C. In some embodiments, the template switching reaction is carried out at a temperature of about 50°C. In some embodiments, the template switching reaction is carried out at a temperature of about 51°C. In some embodiments, the template switching reaction is carried out at a temperature of about 52°C. In some embodiments, the template switching reaction is carried out at a temperature of about 53°C. In some embodiments, the template switching reaction is carried out at a temperature of about 54°C. In some embodiments, the template switching reaction is carried out at a temperature of about 55°C. In some embodiments, the template switching reaction is carried out at a temperature of about 56°C. In some embodiments, the template switching reaction is carried out at a temperature of about 57°C. In some embodiments, the template switching reaction is carried out at a temperature of about 58°C. In some embodiments, the template switching reaction is carried out at a temperature of about 59°C. In some embodiments, the template switching reaction is carried out at a temperature of about 60°C. In some embodiments, the template switching reaction is carried out at a temperature of about 61 °C. In some embodiments, the template switching reaction is carried out at a temperature of about 62°C. In some embodiments, the template switching reaction is carried out at a temperature of about 63 °C. In some embodiments, the template switching reaction is carried out at a temperature of about 64°C. In some embodiments, the template switching reaction is carried out at a temperature of about 65°C. In some embodiments, the template switching reaction is carried out at a temperature of about 66°C. In some embodiments, the template switching reaction is carried out at a temperature of about 67°C. In some embodiments, the template switching reaction is carried out at a temperature of about 68°C. In some embodiments, the template switching reaction is carried out at a temperature of about 69°C. In some embodiments, the template switching reaction is carried out at a temperature of about 70°C. In some embodiments, the templateswitching reaction is carried out at a temperature of about 71 °C. In some embodiments, the template switching reaction is carried out at a temperature of about 72°C. In some embodiments, the template switching reaction is carried out at a temperature of about 73°C. In some embodiments, the template switching reaction is carried out at a temperature of about 74°C. In some embodiments, the template switching reaction is carried out at a temperature of about 75°C. In some embodiments, the template switching reaction is carried out at a temperature greater than 75°C. In some embodiments, the template switching reaction mixture is stored at 4 °C following completion of the template switching reaction.

[0131] In some embodiments, the reverse transcription reaction mixture is incubated at a temperature that allows the polymerase to reverse transcribe the target RNA, e.g., a temperature that permits reverse transcriptase activity of the polymerase. For example, the reverse transcription reaction mixture may be incubated at a temperature of about 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C ,52°C, 53°C, 54°C, 55°C, or greater than 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C or greater. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of less than 4°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 5°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 6°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 7°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 8°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 9°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 10°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 11 °C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 12°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 13 °C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 14°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about15°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 16°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 17°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 18°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 19°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 20°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 21 °C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 22°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 23 °C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 24°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 25°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 26°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 27°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 28°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 29°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 30°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 31 °C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 32°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 33°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 34°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 35°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 36°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 37°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 38°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 39°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 40°C. In some embodiments, the reverse transcription reaction mixture is incubated at atemperature of about 41 °C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 42°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 43 °C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 44°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 45°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 46°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 47°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 48°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 49°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 50°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 51 °C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 52°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 53 °C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 54°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 55°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 56°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 57°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 58°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 59°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 60°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 61 °C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 62°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 63 °C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 64°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 65°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 66°C. In some embodiments, the reverse transcription reaction mixture isincubated at a temperature of about 67°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 68°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 69°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 70°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 71 °C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 72°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 73 °C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 74°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature of about 75°C. In some embodiments, the reverse transcription reaction mixture is incubated at a temperature greater than 75 °C.

[0132] In some embodiments, the reverse transcription reaction is incubated for an amount of time sufficient for cDNAto by synthesized, e.g., incubated for about 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, or 150 minutes or more. In some embodiments, the reverse transcription reaction is incubated for about 1 minute. In some embodiments, the reverse transcription reaction is incubated for about 2 minutes. In some embodiments, the reverse transcription reaction is incubated for about 5 minutes. In some embodiments, the reverse transcription reaction is incubated for about 10 minutes. In some embodiments, the reverse transcription reaction is incubated for about 20 minutes. In some embodiments, the reverse transcription reaction is incubated for about 30 minutes. In some embodiments, the reverse transcription reaction is incubated for about 40 minutes. In some embodiments, the reverse transcription reaction is incubated for about 50 minutes. In some embodiments, the reverse transcription reaction is incubated for about 60 minutes. In some embodiments, the reverse transcription reaction is incubated for about 70 minutes. In some embodiments, the reverse transcription reaction is incubated for about 80 minutes. In some embodiments, the reverse transcription reaction is incubated for about 90 minutes. In some embodiments, the reverse transcription reaction is incubated for about 100 minutes. In some embodiments, the reverse transcription reaction is incubated for about 110 minutes. In some embodiments, the reverse transcription reaction isincubated for about 120 minutes. In some embodiments, the reverse transcription reaction is incubated for about 130 minutes. In some embodiments, the reverse transcription reaction is incubated for about 140 minutes. In some embodiments, the reverse transcription reaction is incubated for about 150 minutes. In some embodiments, the reverse transcription reaction is incubated for greater than 150 minutes.

[0133] In some embodiments, the template switching reaction is incubated for an amount of time sufficient for template switching to occur, e.g., incubated for about 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, or 150 minutes or more. In some embodiments, the template switching reaction is incubated for about 1 minute. In some embodiments, the template switching reaction is incubated for about 2 minutes. In some embodiments, the template switching reaction is incubated for about 5 minutes. In some embodiments, the template switching reaction is incubated for about 10 minutes. In some embodiments, the template switching reaction is incubated for about 20 minutes. In some embodiments, the template switching reaction is incubated for about 30 minutes. In some embodiments, the template switching reaction is incubated for about 40 minutes. In some embodiments, the template switching reaction is incubated for about 50 minutes. In some embodiments, the template switching reaction is incubated for about 60 minutes. In some embodiments, the template switching reaction is incubated for about 70 minutes. In some embodiments, the template switching reaction is incubated for about 80 minutes. In some embodiments, the template switching reaction is incubated for about 90 minutes. In some embodiments, the template switching reaction is incubated for about 100 minutes. In some embodiments, the template switching reaction is incubated for about 110 minutes. In some embodiments, the template switching reaction is incubated for about 120 minutes. In some embodiments, the template switching reaction is incubated for about 130 minutes. In some embodiments, the template switching reaction is incubated for about 140 minutes. In some embodiments, the template switching reaction is incubated for about 150 minutes. In some embodiments, the template switching reaction is incubated for more than 150 minutes.Methods for Enhancing cDNA Synthesis

[0134] The methods described herein enhance cDNA synthesis by a polymerase, e.g., a reverse transcriptase. Enhancing cDNA synthesis may comprise increasing cDNA yield, increasing cDNA length, or reducing off-target products. Separating the reverse transcription and template switching activities of a reverse transcriptase into individual reactions, e.g., allowing reverse transcription to complete before providing a template switching oligonucleotide, may increase cDNA yield, increase cDNA length, and / or reduce off-target products.Increasing cDNA length

[0135] In one aspect, the methods described herein improve synthesis of cDNA libraries from RNA templates, e.g., total cellular RNA. Total cellular RNA can be reverse transcribed into a cDNA library to be used for methods of measuring global gene expression in a cell, e.g., RNA- seq. The accuracy of methods for measuring gene expression in a cell is in part dependent on synthesis and sequencing of full-length cDNAs, e.g., cDNAs that are complementary to the entire template RNA from the 5’ terminus to the 3’ terminus. Synthesis of cDNA begins at the 3’ end of the template RNA and proceeds to the 5’ end of the RNA. Current methods known in the art are limited by incomplete synthesis and sequencing of full-length cDNAs, that is, the 5’ end of the template RNA may not be reverse transcribed. Further, sequencing of the cDNA may require or be facilitated by the presence of barcode sequences covalently linked to the 3’ or 5’ ends of the cDNA. Polymerases having template switching activity, e.g., capable of performing non-templated nucleotide addition and template switching, are known in the art to improve synthesis and sequencing of full-length cDNAs. Methods known in the art for synthesizing cDNA using reverse transcription and template switching allow these activities to be performed concurrently, e.g., the reverse transcription reaction components and the template switching reaction components are provided in the reaction mixture simultaneously.

[0136] In some embodiments, separating the reverse transcription reaction and template switching reactions into individual steps improves the preparation of cDNA libraries, e.g., increases the length of cDNA synthesized from target RNA. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of full-length cDNAs, e.g., cDNAs that are complementary to the entire template RNA from the 5’ terminus to the 3’ terminus. In some embodiments, separating the reversetranscription and template switching reactions into individual steps results in synthesis of cDNAs of about 10 nucleotides (nt) to 100,000 nt in length, e.g., about 400 nt, 800 nt, 1,000 nt, 1,500 nt, 2,000 nt, 2,500 nt, 3,000 nt, 3,500 nt, 4,000 nt, 4,500 nt, 5,000 nt, 5,500 nt, 6,000 nt, 6,500 nt, 7,000, 8,000 nt, 9,000 nt, 10,000 nt, 15,000 nt, 20,000 nt, 30,000 nt, 40,000 nt, 50,000 nt, 60,000 nt, 70,000 nt, 80,000 nt, 90,000 nt, or 100,000 nt or greater in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 400 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 800 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 1,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 1,500 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 2,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 2,500 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 3,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 3,500 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 4,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 4,500 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 5,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 5,500 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 6,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 6,500 nt in length. In some embodiments, separating the reverse transcription and template switching reactions intoindividual steps results in synthesis of cDNAs of about 7,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 8,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 9,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 10,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 15,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 20,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 30,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 40,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 50,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 60,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 70,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 80,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of about 90,000 nt in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in synthesis of cDNAs of greater than 100,000 nt in length.

[0137] In some embodiments, separating the reverse transcription and template switching reactions into individual steps increases the average cDNA length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 1,500 nt to about 10,000 nt in length e.g., about 1,500 nt, 2,000 nt, or 2,500 nt, 3,000 nt, 3,500 nt, 4,000 nt, 4,500 nt, 5,000 nt, 6,000 nt, 7,000 nt, 8,000 nt,9,000 nt, or 10,000 nt or greater in length. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 1,500 nt. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 2,000 nt. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 2,500 nt. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 3,000 nt. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 3,500 nt. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 4,000 nt. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 4,500 nt. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 5,000 nt. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 6,000 nt. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 7,000 nt. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 8,000 nt. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 9,000 nt. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of about 10,000 nt. In some embodiments, separating the reverse transcription and template switching reactions into individual steps results in an average cDNA length of greater than 10,000 nt.

[0138] In some embodiments, separating the reverse transcription reaction and template switching reaction into two individual steps during cDNA synthesis results in longer cDNAs than performing the reverse transcription reaction and template switching reaction concurrently in one step, e.g., results in cDNAs that are 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or greater in length. In someembodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 10% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 20% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 30% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 40% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 50% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 60% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 70% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 80% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 90% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 100% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 150% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 200% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 250% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 300% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 350% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 400% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction asI lltwo individual steps results in cDNAs that are 450% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are 500% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps results in cDNAs that are greater than 500% greater in length. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 10%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 20%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 30%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 40%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 50%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 60%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 70%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 80%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 90%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 100%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 150%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 200%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 250%. In some embodiments, performing thereverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 300%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 350%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 400%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 450%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by 500%. In some embodiments, performing the reverse transcription reaction and template switching reaction as two individual steps increases the average cDNA length in a cDNA library by greater than 500%. In some embodiments, performing the reverse transcription reaction as two individual steps increases the average cDNA length by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900% or more, relative to reverse transcription and template switching reactions that are performed concurrently.

[0139] In some embodiments, contacting a reverse transcription reaction mixture with a template switching reaction mixture, e.g., a template switching oligonucleotide (TSO), additional dATP, PEG4000, and / or additional polymerase, after a period of time, e.g., after the reverse transcription reaction is allowed to proceed for a period of time, increases cDNA length, e.g., by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900% or more, relative to reverse transcription and template switching reactions that are performed concurrently. The period of time may comprise about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more minutes. The reverse transcription may or may not reach completion prior to the contacting with a template switching reaction mixture. In some embodiments, contacting a reverse transcription reaction mixture with a template switching reaction mixture after the reverse transcription reaction has proceeded for a period of time increases cDNA length by about 10- 900%, relative to reverse transcription and template switching reactions that are performed concurrently. In some embodiments, contacting a reverse transcription reaction mixture with a template switching reaction mixture after the reverse transcription reaction has proceeded for aperiod of time increases cDNA length by about 50-500%, relative to reverse transcription and template switching reactions that are performed concurrently. In some embodiments, contacting a reverse transcription reaction mixture with a template swit...

Claims

CLAIMS1. A method of enhancing complementary DNA (cDNA) synthesis, e.g., from an RNA template, by a polymerase having reverse transcriptase and template switching activity, e.g., a reverse transcriptase, the method comprising:(a) preparing a first reaction mixture comprising:(i) a target RNA, e.g., a plurality of target RNAs;(ii) a reverse transcription primer;(iii) a deoxyribonucleotide triphosphate (dNTP) solution; and(iv) a polymerase, e.g., a reverse transcriptase, and(b) allowing the first reaction mixture to incubate for a time period;(c) preparing a second reaction mixture, comprising(v) the first reaction mixture after step (b); and(vi) a template switching oligonucleotide; thereby enhancing synthesis of cDNA.

2. The method of claim 1, wherein enhancing comprises improving the efficiency of cDNA synthesis by the polymerase and / or improving the yield of cDNA synthesis by the polymerase.

3. The method of claim 1, wherein the second reaction mixture further comprises:(vii) a deoxyadenosine triphosphate (dATP) solution;(viii) a polyethylene glycol; and(ix) a polymerase, e.g., a reverse transcriptase.

4. The method of claim 1, wherein the first reaction mixture has greater ionic strength than the second reaction mixture.

5. The method of claim 1, wherein the first reaction mixture comprises an equimolar mixture of deoxyribonucleotides (dNTPs), e.g., an equimolar mixture of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), and deoxyguanosine triphosphate (dGTP).

6. The method of claim 1, wherein the second reaction mixture has lower ionic strength than the first reaction mixture.

7. The method of claim 1, wherein the second reaction mixture comprises a greater amount of deoxy adenosine triphosphate (dATP) relative to deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), or deoxyguanosine triphosphate (dGTP), e.g., a 2 times, 3 times, 4 times or greater amount of dATP relative to dCTP, dTTP, or dGTP.

8. The method of claim 1, wherein the enhancing cDNA synthesis comprises increasing the length of the cDNA synthesized by the polymerase, e.g., increasing the length of the cDNAby 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more.

9. The method of claim 1, wherein the cDNA synthesized is full-length cDNA, e.g., cDNA that is complementary to an entire target RNA sequence, e.g., from the 5’ terminus of the target RNAto the 3’ terminus of the target RNA.

10. The method of claim 9, wherein the cDNA is between about 10 nucleotides (nt) and 100,000 nt in length, e.g., about 500 nt, 1000 nt, 10,000 nts, 50,000 and 100,000 nt in length.

11. The method of claim 10, wherein the average cDNA length is between about 1,500 nt and 2,500 nt in length, e.g., about 1,500 nt, 2,000 nt, 2,500 nt, 3,000 nt, 3,500 nt, 4,000 nt, 4,500 nt, 5,000 nt, or greater than 5,000 nt in length.

12. The method of claim 1, wherein the cDNA is synthesized from total cellular RNAto generate a cDNA library.

13. The method of claim 1, wherein the reverse transcription reaction in the first step is incubated for at least 1 minute before being contacted with the template switching reaction components in the second step, e.g., incubated for at least 1 minute, 3 minutes, 5 minutes, 10minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, or 150 minutes or more.

14. The method of claim 1, wherein the level of the target RNA in the mixture is between about 1 pg to about 0.1 pg.

15. The method of claim 1, wherein the polymerase is a DNA polymerase or an RNA polymerase.

16. The method of claim 1, wherein the polymerase is a reverse transcriptase.

17. The method of claim 16, wherein the reverse transcriptase is derived from a virus, an intron, a telomerase, a retrotransposon, a polymerase with reverse transcriptase activity, or an engineered polymerase with reverse transcriptase activity.

18. The method of claim 16, wherein the reverse transcriptase is a group II intron reverse transcriptase, a telomerase reverse transcriptase, a viral reverse transcriptase or a retroviral reverse transcriptase.

19. The method of claim 16, wherein the reverse transcriptase comprises MarathonRT™, UltraMarathonRT®, Induro®, Maxima™ H Minus, SuperScript™ II, SuperScript™ III, SuperScript™ IV, PrimeScripf™, Transcriptor, GoScript™, ProtoScript® II, SMARTScribe™, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, Bombyx Mori RT, telomerase RT, TGIRT™, or a fragment, variant, mutant, or derivative thereof.

20. The method of claim 16, wherein the reverse transcriptase comprises any one of SEQ IDNOs: 1-15.21 . The method of claim 16, wherein the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to any one of SEQ ID NOs: 1-15, e.g., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to any one of SEQ ID NOs: 1-15.

22. The method of claim 16, wherein the reverse transcriptase consists of any one of SEQ ID NOs: 1-15.

23. The method of claim 16, wherein the reverse transcriptase comprises MarathonRT™, or a fragment, variant, mutant, or derivative thereof.

24. The method of claim 16, wherein the reverse transcriptase comprises SEQ ID NO:1 or SEQ ID NO:2.

25. The method of claim 16, wherein the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to any one of SEQ ID NOs: 1 or 2, e.g., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to any one of SEQ ID NOs: 1 or 2.

26. The method of claim 16, wherein the reverse transcriptase consists of SEQ ID NO: 1 or SEQ ID NO:2.

27. The method of claim 16, wherein the reverse transcriptase comprises UltraMarathonRT®, or a fragment, variant, mutant, or derivative thereof.

28. The method of claim 16, wherein the reverse transcriptase comprises a sequence with about 60% to about 99.9% identity to any one of SEQ ID NOs: 14 or 15, e.g., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater identity to any one of SEQ ID NOs: 14 or 15.

29. The method of claim 16, wherein the reverse transcriptase comprises SEQ ID NO: 14 or SEQ ID NO: 15.

30. The method of claim 16, wherein the reverse transcriptase consists of SEQ ID NO: 14 or SEQ ID NO: 15.

31. The method of claim 1, wherein the reverse transcription primer is between 5 and 500 nucleotides in length.

32. The method of claim 1, wherein the reverse transcription primer is complementary to the 3’ end of the target RNA.

33. The method of claim 1, wherein first reaction mixture further comprises a plurality of input RNA sequences (e.g., non-target RNA).

34. The method of claim 1, further comprising step (b’), wherein the first reaction mixture is separated or purified after step (b).

35. The method of claim 34, wherein the separation or purification comprises filtration, centrifugation, chromatography, or electrophoresis.

36. The method of of claim 1, further comprising step (d) allowing the second reaction mixture to incubate for a time period.

37. The method of claim 36, wherein step (d) occurs after step (c).

38. The method of claim 1, wherein the method comprises:(i) detecting the level, identity or concentration of a target RNA;(ii) increasing the signal to noise ratio of a target RNA; and / or(iii) increasing the processivity of the reverse transcriptase reaction, compared to a reference standard.

39. A method of enhancing complementary DNA (cDNA) synthesis, e.g., from an RNA template, by a polymerase having reverse transcriptase and template switching activity, e.g., a reverse transcriptase, the method comprising:(a) preparing a first reaction mixture comprising:(i) a target RNA, e.g., a plurality of target RNAs;(ii) a reverse transcription primer;(iii) a deoxyribonucleotide triphosphate (dNTP) solution; and(iv) a polymerase, e.g., a reverse transcriptase, and(b) allowing the first reaction mixture to incubate for a time period;(c) isolating the cDNA product from the reaction mixture;(d) preparing a second reaction mixture, comprising(v) the cDNA product after step (c);(vi) a deoxyribonucleotide triphosphate (dNTP) solution;(vii) a polymerase, e.g., a reverse transcriptase, and(viii) a template switching oligonucleotide;(e) allowing the second reaction mixture to incubate for a time period, thereby enhancing synthesis of cDNA.

40. A kit for enhancing complementary DNA (cDNA) synthesis, e.g., from an RNA template, comprising:(i) a polymerase having reverse transcriptase and template switching activity (e.g., a reverse transcriptase, e.g., MarathonRT™);(ii) a deoxyribonucleotide triphosphate (dNTP) solution; and(iii) a buffer solution; and(iv) a template switching oligonucleotide.

41. The kit of claim 40, further comprising a reverse transcription primer.

42. The kit of claim 40, further comprising a deoxyadenosine triphosphate (dATP) solution.

43. The kit of claim 40, wherein (iv) is provided separately from (i)-(iii).

44. The kit of claim 40, wherein the dATP is provided together with the template switching oligonucleotide.

45. The kit of claim 40, wherein the polymerase comprises MarathonRT™, UltraMarathonRT®, Induro®, Maxima™ H minus, SuperScript™ II, SuperScript™ III, SuperScript™ IV, PrimeScript™, Transcriptor, GoScript™, ProtoScript® II, SMARTScribe™, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, TGIRT™, or a fragment, variant, mutant, or derivative thereof.

46. The kit of claim 40, wherein the polymerase comprises a group II intron RT, e.g., UltraMarathonRT®.

47. The kit of claim 40, wherein the template switching oligonucleotide is between 5 and 500 nucleotides in length.

48. The kit of claim 41, wherein the reverse transcription primer is between 5 and 500 nucleotides in length.

49. The kit of claim 40, wherein the kit is useful for:(i) acquiring a value for the presence of a target ribonucleic acid (RNA) in a mixture;(ii) detecting the level, identity or concentration of a target RNA;(iii) increasing the signal to noise ratio of a target RNA;(iv) preparing a library for the target RNA templates;(v) increasing the processivity of the reverse transcriptase reaction; and / or(vi) increasing the template switching efficiency of the reverse transcriptase reaction.

Images