mRNA sequencing libraries, construction methods, and reagent kits

Abstract

This invention relates to the field of biological sequencing, specifically to mRNA sequencing libraries, construction methods, and kits. The cell lysis buffer used in the mRNA sequencing library construction method provided by this invention contains a high concentration of Triton X-100, which can completely dissolve trace amounts of cell membranes and nuclear membranes, releasing all free and bound RNA.

Description

Technical Field

[0001] This disclosure relates to the field of biological sequencing, specifically to mRNA sequencing libraries, construction methods, and reagent kits. Background Technology

[0002] Transcriptome sequencing is an important tool for studying gene expression and its regulation. In life science research, RNA extraction and transcriptome analysis from low-sample starting materials always present significant challenges.

[0003] In experiments involving small amounts of primary cells, cultured cells, tumor tissue cells, or plant cells, 96-well or 384-well plates are typically used, with each well containing hundreds to tens of thousands of cells. At such low cell volumes, the amount of RNA that can be extracted using conventional methods is very small, often insufficient to meet the starting quantity requirements for subsequent large-scale gene expression assays.

[0004] For such small sample sizes, the usual practice is to first reverse transcribe RNA into cDNA, and then amplify the cDNA to obtain sufficient cDNA for constructing sequencing libraries. Traditional RNA extraction and purification steps (such as nucleic acid precipitation and elution) can lead to severe absolute RNA loss when dealing with small amounts of cells due to physical adsorption to the tube walls and multiple centrifugation transfers. The loss of low-abundance mRNA is particularly severe, which can cause deviations in gene expression quantification and even directly lead to library construction failure or extremely low gene detection rates.

[0005] While existing extraction-free crude lysis direct amplification techniques avoid RNA loss during multiple transfer processes, the crude lysis buffer contains a large amount of genomic DNA, proteins, lipids, and abundant ribosomal RNA (rRNA). These impurities not only non-specifically consume amplification primers in subsequent reactions but also severely interfere with the specific capture or amplification of mRNA, resulting in a high residual rate of rRNA in the final sequencing data and reducing the amount of effective sequencing data.

[0006] In addition, the throughput of sample detection is also limited, making it difficult to meet the demand for parallel detection of multiple samples and simultaneous detection of the expression levels of tens of thousands of genes.

[0007] Therefore, there is an urgent need in this field for a method specifically designed for RNA extraction and high-throughput transcriptome sequencing of a small number of cell or tissue samples, in order to obtain a high-quality library that meets the requirements of transcriptome sequencing under limited cell or tissue quantity conditions, and to achieve parallel detection of tens of thousands of gene expression levels in multiple samples. Summary of the Invention

[0008] The purpose of this disclosure is to provide a method for constructing an mRNA sequencing library. This method uses cell lysis buffer containing a high concentration of Triton X-100, which increases the amount of mRNA extracted from cell samples, thereby significantly reducing the lower limit of the applicable range of the number of live cells in the sample. This enables the construction method provided by this disclosure to achieve the goal of constructing a reliable mRNA sequencing library from a low sample size.

[0009] Another objective of this disclosure is to adjust the incubation temperature after cell lysis in the aforementioned mRNA sequencing library construction method to a relatively high level, thereby further increasing the amount of mRNA extracted from cell samples.

[0010] This disclosure provides a method for constructing an mRNA sequencing library, including: Cell samples containing mRNA were lysed using cell lysis buffer. mRNA was extracted from the cell lysis products by incubation at 20–75°C using an mRNA capture agent. The obtained mRNA was eluted and fragmented. First-strand cDNA was synthesized using the fragmented mRNA as a template, followed by second-strand cDNA synthesis to obtain double-stranded cDNA. The double-stranded cDNA underwent end repair and A-tailing, was then ligated with sequencing adapters, and amplified by PCR to obtain a sequencing library. The cell lysis buffer consisted of 20–60 U / μL of RNase inhibitor and 5%–12% (w / v) of Triton X-100. The cell sample contained 1000–150000 live cells.

[0011] In an optional embodiment, the cell lysis buffer contains 30-50 U / μL of RNase inhibitor.

[0012] In an optional embodiment, the cell lysis buffer contains 8% to 12% (w / v) of Triton X-100.

[0013] In an optional embodiment, the cell lysis buffer consists of 20-60 U / μL of an RNase inhibitor and 10% (w / v) of Triton X-100.

[0014] In an optional embodiment, the cell lysis buffer consists of 40 U / μL of an RNase inhibitor and 10% (w / v) of Triton X-100.

[0015] In an optional embodiment, the incubation temperature is 22~72°C.

[0016] In an optional embodiment, the incubation temperature is 72°C.

[0017] In an optional embodiment, the mRNA capturing agent comprises a solid-phase carrier incorporating an mRNA capturing probe.

[0018] In an optional embodiment, the solid support is selected from microspheres, microparticles, chip films, and container inner walls.

[0019] In an optional embodiment, the mRNA capturing agent is a magnetic bead bound to oligodeoxythymidine nucleotides.

[0020] In an optional embodiment, the cell sample contains 1,000 to 5,000 live cells.

[0021] In an optional implementation, the construction method includes: Cell samples containing mRNA were lysed using cell lysis buffer. mRNA was extracted from the cell lysis products by incubation at 72°C using magnetic beads conjugated with oligodeoxythymidine nucleotides. The mRNA was eluted and fragmented. First-strand cDNA was synthesized using the fragmented mRNA as a template, followed by second-strand cDNA synthesis to obtain double-stranded cDNA. The double-stranded cDNA underwent end repair and A-tailing, was then ligated with sequencing adapters, and amplified by PCR to obtain a sequencing library. The cell lysis buffer consisted of 40 U / μL of RNase inhibitor and 10% (w / v) Triton X-100. The cell sample contained 1000–5000 live cells.

[0022] Another aspect of this disclosure provides a kit for constructing an mRNA sequencing library, the kit comprising a cell lysis buffer consisting of 20-60 U / μL of an RNase inhibitor and 5%-12% (w / v) of Triton X-100.

[0023] In an optional embodiment, the cell lysis buffer contains 30-50 U / μL of RNase inhibitor.

[0024] In an optional embodiment, the cell lysis buffer contains 8% to 12% (w / v) of Triton X-100.

[0025] In an optional embodiment, the cell lysis buffer consists of 20-60 U / μL of an RNase inhibitor and 10% (w / v) of Triton X-100.

[0026] In an optional embodiment, the cell lysis buffer consists of 40 U / μL of an RNase inhibitor and 10% (w / v) of Triton X-100.

[0027] Another aspect of this disclosure provides a method for detecting the mRNA content in a cell sample, the method comprising constructing an mRNA sequencing library of the cell sample using the construction method described in any of the foregoing embodiments, and quantitatively detecting the mRNA content in the mRNA sequencing library.

[0028] This disclosure also provides a method for transcriptome sequencing of cell samples, wherein the transcriptome sequencing method uses the construction method or the kit described in any of the foregoing embodiments to construct an mRNA sequencing library and then sequences it.

[0029] The cell lysis buffer used in the method for constructing the mRNA sequencing library disclosed herein contains a high concentration of Triton X-100, which can completely dissolve the cell membrane and nuclear membrane of trace cells, releasing all free and bound RNA.

[0030] On the other hand, during the construction of mRNA sequencing libraries, intentionally increasing the incubation temperature of cell lysis products can further increase the amount of RNA released. In addition, high concentrations of Triton X-100 and high temperatures can denature endogenous RNase and other RNA-binding proteins, fully unraveling the complex secondary structure of RNA and exposing the poly-A tail of mRNA to the greatest extent, thereby further increasing the amount of mRNA extracted.

[0031] On the other hand, the complete lysis process and magnetic bead binding process in the mRNA sequencing library construction method provided in this disclosure are both completed in the same tube. In some embodiments, the separation step after lysis can be omitted, which significantly reduces the transfer loss during the mRNA extraction process. Attached Figure Description

[0032] Figure 1 The results of the final library DNA fragment size distribution detection in Example 1; Figure 2 This is the result of the DNA fragment size distribution detection in the final library of Example 1; Figure 3 This is the result of the DNA fragment size distribution detection in the final library of Example 2; Figure 4 The results show the number of effectively expressed genes in the sequencing libraries obtained by the three methods in Example 1, Control Example 1, and Control Example 2. Figure 5 The results of Pearson correlation detection in sequencing libraries obtained by the three methods in Example 1, Control Example 1, and Control Example 2 are shown. Figure 6 To verify the results of detecting the number of effectively expressed genes in the sequencing libraries obtained by the three methods in Example 1; Figure 7 To verify the Pearson correlation detection results in the sequencing libraries obtained by the three methods in Example 1. Detailed Implementation

[0033] Based on the above content of this disclosure, and in accordance with common technical knowledge and practices in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical ideas of this disclosure.

[0034] I. Definition Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0035] Throughout this specification and the appended claims, unless the context otherwise requires, the words “comprise” and variations such as “comprises” and “comprising” are to be understood as implying the inclusion of the stated member, integer, or step, or a group of members, integers, or steps, but do not exclude any other member, integer, or step or group of members, integers, or steps. However, in some embodiments, such other members, integers, or steps or groups of members, integers, or steps may be excluded, i.e., the subject matter is to include the stated member, integer, or step or group of members, integers, or steps.

[0036] Unless otherwise indicated herein or clearly contradicted by the context, the terms “a / an” and “described” and similar references used in the context of describing this disclosure (especially in the context of the claims) shall be considered to cover both the singular and plural. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually enumerated herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “such”) provided herein is intended only to better illustrate this disclosure and does not limit the scope of this disclosure as otherwise claimed. All language in this specification should not be construed as indicating that any unclaimed element is necessary for the practice of this disclosure.

[0037] Several documents are referenced in full in the main body of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's instructions, guidance, etc.) is incorporated herein by reference in its entirety.

[0038] The term "about" refers to a range of acceptable error for a particular value as determined by those skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular measurement, result, or implementation, unless otherwise expressly stated in the embodiments or elsewhere in the specification, "about" means within one standard deviation, or up to 5% (whichever is greater), according to convention in the art.

[0039] The mRNA sequencing library described in this article refers to a collection of nucleic acid molecules that are prepared using messenger RNA (mRNA) as the starting or target nucleic acid and can be used for nucleic acid sequencing. The nucleic acid molecules in the library typically have adapter sequences compatible with the sequencing platform at both ends, and may optionally carry index sequences for distinguishing samples and / or molecular tags for correcting amplification bias.

[0040] As used in this article, "cell lysis buffer" refers to a class of chemical reagents used to disrupt cell membranes and release intracellular contents such as proteins, nucleic acids, and metabolites. The lysis process typically involves physical or chemical methods, while cell lysis buffers provide a suitable chemical environment to ensure the stability of cell contents and the smooth execution of subsequent experiments. Cell lysis buffers typically contain lysing agents (e.g., surfactants) and RNA protectants.

[0041] The cell sample described herein refers to a sample containing one or more cells, or derived from a biological fluid sample, such as primary cells, cultured cells, tumor tissue cells, organoid cells, and plant cells. The cell sample may be in the form of a cell suspension or cell lysate and typically contains mRNA. As a non-limiting example, the biological fluid sample includes blood, plasma, serum, sweat, tears, sputum, urine, ear flow, lymph, interstitial fluid, saliva, cerebrospinal fluid, vomitus, bone marrow suspension, vaginal flow, cervical lavage fluid, cerebrospinal fluid, ascites, breast milk, respiratory secretions, intestinal and genitourinary tract fluid, amniotic fluid, and samples separated from leukocytes. In some embodiments, the source sample is a sample readily obtainable through non-invasive procedures, such as blood, plasma, serum, sweat, tears, sputum, urine, ear flow, and saliva. In some embodiments, the cell sample is derived from peripheral blood, or a biological sample such as plasma or serum that has been further separated. In other embodiments, the cell sample is cells from a swab or smear, a biopsy cell sample, or a cell culture. In yet another embodiment, the cell sample may also be a mixture of two or more cell samples, such as a biological sample comprising two or more of the following: a biological fluid sample, a tissue sample, and a cell culture sample.

[0042] The mRNA capture agent described herein refers to a substance or moiety capable of specifically binding to and enriching mRNA. The mRNA capture agent typically achieves capture through complementary hybridization with the poly(A) tail or specific fragment of mRNA. For example, in some specific embodiments, the mRNA capture agent comprises a solid-phase carrier coupled with oligo-dT; the solid-phase carrier can be selected from microspheres, microparticles, chip membranes, and container inner walls, such as magnetic beads.

[0043] The fragmentation process described in this article refers to the process of breaking nucleic acid molecules into shorter fragments, which can be achieved through physical methods (such as ultrasound or heating), chemical methods (such as the breaking of divalent metal ions under heating conditions), or enzymatic methods (such as endonucleases or transposases).

[0044] The term "reverse transcription" as used in this article refers to the process by which complementary DNA strands (cDNA) are synthesized from single-stranded RNA as a template under the action of reverse transcriptase, following the base pairing principle (AT, CG, UA), ultimately forming double-stranded DNA. This process is a special form of DNA biosynthesis, breaking through the traditional unidirectional flow pattern of genetic information from DNA to RNA and then to protein.

[0045] This document discloses certain embodiments in which feasible reverse transcription methods include Smart-seq, Smart-seq2, Oligo(dT) primers, and random primers. Among these, Smart-seq reverse transcription is a single-cell transcriptome sequencing technology developed by scientists in the United States and Sweden in 2012, designed to capture the full-length sequence of mRNA. Its core technology involves using Moloney mouse leukemia virus reverse transcriptase (MMLVRT) to add a non-templated cytosine (C) to the 3' end of cDNA via template switching, followed by the synthesis of double-stranded cDNA using primer 2 (containing a universal sequence), and subsequent PCR amplification. Its advantages include full-length transcript coverage, suitability for alternative splicing analysis, and single nucleotide polymorphism (SNP) detection.

[0046] The Smart-seq2 reverse transcription method is an improved version of Smart-seq (2013), optimizing reverse transcription and amplification efficiency. It uses oligo(dT)VN primers to bind to the poly(A) tail of mRNA, and through the template-changing activity of MMLVRT, adds a C residue to the 3' end of the first strand of cDNA, combining with locked nucleic acid (LNA)-modified template-changing oligonucleotides (TSOs) to synthesize the second strand. Its advantages include higher sensitivity and cDNA integrity, making it suitable for low-quality RNA samples.

[0047] The Oligo(dT) primer method uses primers composed of 12-20 deoxythymidine nucleotides that bind to the poly(A) tail of mRNA through complementary pairing, initiating reverse transcription. It is suitable for the synthesis of full-length cDNA in eukaryotes. Its advantages include high specificity (targeting only mRNA).

[0048] Random priming uses a primer set composed of random hexamers (N6) that can bind to any RNA sequence (including mRNA, rRNA, and tRNA) to initiate the synthesis of short cDNA fragments. Its characteristics include low specificity, making it suitable for templates that degrade RNA or complex secondary structures.

[0049] cDNA and cDNA synthesis: In this article, "cDNA" (complementary DNA) refers to complementary deoxyribonucleic acid synthesized by reverse transcription using RNA as a template; "cDNA synthesis" refers to the process of generating cDNA, including the synthesis of first-strand cDNA using RNA as a template under the action of reverse transcriptase, and the synthesis of second-strand cDNA using first-strand cDNA as a template to obtain double-stranded cDNA.

[0050] End repair, as described in this article, refers to the process of treating the ends of double-stranded DNA to form ends suitable for subsequent ligation. This typically includes filling in uneven ends to blunt ends and / or phosphorylating the 5' end.

[0051] The A-tailing (dA-tailing / A-tailing) described in this article refers to the process of adding a single deoxyadenosine nucleotide (A) to the 3' end of double-stranded DNA in order to ligate it to a adapter with a corresponding thymidine (T) overhang.

[0052] The PCR amplification described herein refers to the process of exponentially amplifying a target nucleic acid sequence in vitro through polymerase chain reaction (PCR) with the help of primers and DNA polymerase; optionally, adapter sequences and / or index sequences may be introduced into the product during this process.

[0053] The RNase inhibitors mentioned in this article refer to substances that can inhibit the activity of ribonuclease (RNase) and thus reduce RNA degradation. These include protein RNase inhibitors and reducing agents (such as dithiothreitol or β-mercaptoethanol) that inactivate RNase by breaking its disulfide bonds.

[0054] The kits described herein refer to a collection of one or more reagents and / or components provided in combination for a specific purpose, and may optionally include a carrier (e.g., a microplate), a control, and / or instructions for use. The components in the kits may be aliquoted.

[0055] II. Detailed Description of the Invention Cell lysate This disclosure provides a cell lysis buffer for constructing mRNA sequencing libraries, the lysis buffer being used to lyse live cells to fully obtain free and bound mRNA in the cytoplasm and nucleus.

[0056] In some specific embodiments, the cell lysis buffer consists of an RNA protectant, a lysis agent, and water.

[0057] It is understood that the solvent of the cell lysis buffer is water. Those skilled in the art can select water solvents that have been treated by different methods commonly used in various biological experiments as the solvent of the cell lysis buffer in this disclosure, including but not limited to distilled water, nanofiltration water, deionized water, ultrapure water, sterile water and DEPC treated water, according to actual experimental conditions.

[0058] Distilled water refers to water obtained by removing most of the inorganic salts and non-volatile impurities through heating, vaporization, and condensation. It can be further classified into single-distilled, double-distilled, and triple-distilled water.

[0059] The nanofiltration water is water filtered through a nanofiltration membrane, and the pore size of the nanofiltration membrane is typically 1 to 5 nanometers.

[0060] The deionized water refers to water that has been sequentially filtered through activated carbon, treated with cation exchange resin, treated with anion exchange resin, and treated with mixed bed resin to adsorb and remove cations and anions from the water.

[0061] The ultrapure water is obtained after undergoing multi-stage treatment to significantly remove all impurities. The conventional treatment steps include, but are not limited to, pretreatment (polypropylene meltblown filter element and activated carbon adsorption), reverse osmosis membrane treatment, electro-desalination treatment, ion exchange mixed bed treatment, and ultrafiltration treatment.

[0062] The sterile water refers to water obtained by high-pressure steam sterilization (121°C, 20 min) or sterile filtration (e.g., negative pressure filtration using a 0.22 μm water-based filter membrane).

[0063] The DEPC (diethyl pyrocarbonate) treated water refers to water that has been treated with DEPC, in which the RNA-degrading enzymes in the water are covalently modified by DEPC to prevent the degradation of subsequently added RNA.

[0064] It is understandable that the different water treatment methods commonly used above can be used in combination. For example, in some embodiments, the water used to prepare cell lysate is first distilled and then treated with DEPC.

[0065] In some specific embodiments, the solvent for the cell lysis buffer is nanofiltration water.

[0066] In some specific embodiments, the cell lysis buffer of this disclosure contains 20-60 U / μL of RNase inhibitor as an RNA protectant; the concentration may be 30-50 U / μL, including but not limited to 20 U / μL, 25 U / μL, 30 U / μL, 35 U / μL, 40 U / μL, 45 U / μL, 50 U / μL, 55 U / μL or 60 U / μL.

[0067] In some specific embodiments, the cell lysis buffer described in this disclosure contains 5% to 12% (w / v) Triton X-100 as a lysis agent, which may be selected as 8% to 12% (w / v), including but not limited to 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), 10% (w / v), 11% (w / v), or 12% (w / v). In some specific embodiments, the cell lysis buffer described in this disclosure consists of 20-60 U / μL of an RNase inhibitor and 10% (w / v) of Triton X-100.

[0068] In some specific embodiments, the cell lysis buffer described in this disclosure consists of 40 U / μL of an RNase inhibitor and 10% (w / v) of Triton X-100.

[0069] This disclosure selects a relatively high concentration of Triton X-100 as a cell lysis agent, which can lyse cell membranes and nuclear membranes to a large extent, fully releasing free and bound RNA. Furthermore, in some embodiments, when the cell lysis products are directly mixed with the mRNA scavenging agent, the high concentration of Triton X-100 can act as a "blocking agent" and / or a "washing agent," effectively preventing the non-specific adsorption of impurities such as rRNA, large genomic fragments, and denatured proteins on the surface of the mRNA scavenging agent through steric hindrance and strong interfacial activity. For example, when using magnetic beads conjugated with oligodeoxythymidine nucleotides (oligo-dT) as the mRNA scavenging agent, the high concentration of Triton X-100 can prevent the non-specific binding of impurities such as rRNA, large genomic fragments, and denatured proteins to the surface of the magnetic beads, ensuring that the residual amount of impurities such as rRNA is controlled to an acceptable level even without centrifugation purification of the cell lysis products.

[0070] mRNA sequencing library construction methods This disclosure provides a method for constructing mRNA sequencing libraries. By improving the cell lysis process, it is made applicable to the construction of sequencing libraries from samples with low cell content, expanding the range of cell content in samples that can be used for sequencing library construction. This allows the method to be further applied to sequencing analysis where it is difficult to obtain samples of the target expressed gene, and thus to early screening in the early stages of disease development where the amount of the target expressed gene is low.

[0071] In some specific embodiments, the method for constructing the mRNA sequencing library described in this disclosure includes: Cell samples containing mRNA were lysed using cell lysis buffer, and mRNA was extracted from the cell lysis products by incubation at 20–75°C using an mRNA capture agent. The mRNA was eluted and then used for library construction. The cell lysis buffer consisted of 20–60 U / μL of RNase inhibitor and 5%–12% (w / v) of Triton X-100. The cell sample contained 1,000–150,000 live cells.

[0072] It should be noted that the cell lysis buffer described in the aforementioned embodiments significantly improves the quality of mRNA extraction. Furthermore, the incubation temperature of the cell lysis product and the mRNA capture agent can be adjusted. Incubation at a relatively high temperature further denatures endogenous RNase and other RNA-binding proteins, fully unwinding the complex secondary structure of RNA and maximizing the exposure of the poly-A tail of mRNA, thereby further increasing the amount of mRNA extracted.

[0073] In some specific embodiments, the cell lysate contains 30-50 U / μL of RNase inhibitor.

[0074] In some specific embodiments, the cell lysate contains 8% to 12% (w / v) of Triton X-100.

[0075] In some specific embodiments, the cell lysis buffer consists of 20-60 U / μL of an RNase inhibitor and 10% (w / v) of Triton X-100.

[0076] In some specific embodiments, the cell lysis buffer consists of 40 U / μL of an RNase inhibitor and 10% (w / v) of Triton X-100.

[0077] In some specific embodiments, the incubation temperature can be 20~75℃, and can be selected from 22~72℃, including but not limited to 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, 32℃, 34℃, 36℃, 38℃, 40℃, 42℃, 44℃, 46℃, 48℃, 50℃, 52℃, 54℃, 56℃, 58℃, 60℃, 62℃, 64℃, 66℃, 68℃, 70℃, 71℃, 72℃, 73℃, 74℃ or 75℃.

[0078] In some specific embodiments, the mRNA capturing agent includes a solid-phase carrier in which an mRNA capturing probe is bound.

[0079] In some specific embodiments, the solid support may be selected from microspheres, microparticles, chip films, and the inner wall of a container.

[0080] In some specific embodiments, the mRNA capture probe may be bound to oligodeoxythymidine nucleotides.

[0081] In some specific embodiments, the mRNA capturing agent is a magnetic bead bound to oligodeoxythymidine nucleotides.

[0082] In some specific embodiments, the number of live cells contained in the cell sample is 1,000 to 150,000, or 1,000 to 5,000, including but not limited to 1,000, 5,000, 15,000, 20,000, 80,000, 100,000, or 150,000.

[0083] It is understandable that, given a high-quality mRNA sample, those skilled in the art can routinely select subsequent library construction steps and methods based on the actual situation.

[0084] In some specific implementations, the database construction step includes the following (a) or (b): (a) The obtained mRNA was fragmented, and the fragmented mRNA was used as a template for reverse transcription to obtain fragmented double-stranded cDNA. After end repair and A-tailing, sequencing adapters were ligated and PCR amplification was performed to obtain sequencing libraries. (b) Using the obtained mRNA as a template, reverse transcription was performed to obtain complete double-stranded cDNA. Tn5 enzyme transposons were added to obtain fragmented double-stranded cDNA. DNA polymerase was used to fill the gaps in the double-stranded cDNA, and PCR amplification was performed to obtain a sequencing library.

[0085] The Tn5 enzyme transposon typically comprises a Tn5 enzyme dimer, each of which binds to a Mosaic End (ME) linker to assemble into a stable enzyme-DNA complex. The Tn5 enzyme transposon is located in Mg... 2+ When activated, it can recognize and cut conserved DNA sequences while introducing ME adapters at the ends.

[0086] In some specific implementations, feasible library construction methods include: first, fragmenting the obtained mRNA; then, using the fragmented mRNA as a template to synthesize a first-strand cDNA; and finally, using the first-strand cDNA as a template to synthesize a second-strand cDNA to obtain double-stranded cDNA; sequentially performing end repair and A-tailing on the double-stranded cDNA; then ligating sequencing adapters; and finally, performing PCR amplification to obtain a sequencing library. This method includes multiple steps such as mRNA fragmentation, synthesis of two cDNA strands, ds-cDNA end processing, and adapter ligation. Those skilled in the art can routinely select these steps according to actual needs, such as purchasing commercially available kits and operating according to the instructions.

[0087] In some specific implementations, feasible library construction methods include: using the obtained mRNA as a template for reverse transcription to obtain complete double-stranded cDNA, adding a Tn5 transposon and Mg... 2+ The buffer solution is used to process the fragmented double-stranded cDNA, and DNA polymerase is used to fill the gaps in the double-stranded cDNA. PCR amplification is then performed to obtain the sequencing library. For these steps, those skilled in the art can make conventional choices according to actual needs, such as purchasing commercially available kits and operating according to the instructions.

[0088] In some specific embodiments, the method for constructing the mRNA sequencing library described in this disclosure includes: Cell samples containing mRNA were lysed using cell lysis buffer. mRNA was extracted from the cell lysis products by incubation at 20–75°C using an mRNA capture agent. The obtained mRNA was eluted and fragmented. First-strand cDNA was synthesized using the fragmented mRNA as a template, followed by second-strand cDNA synthesis to obtain double-stranded cDNA. The double-stranded cDNA underwent end repair and A-tailing, was then ligated with sequencing adapters, and amplified by PCR to obtain a sequencing library. The cell lysis buffer consisted of 20–60 U / μL of RNase inhibitor and 5%–12% (w / v) of Triton X-100. The cell sample contained 1000–150000 live cells.

[0089] On the other hand, the complete lysis process and magnetic bead binding process in the mRNA sequencing library construction method provided in this disclosure are both completed in the same tube. In some embodiments, the centrifugation step after lysis can be omitted, which significantly reduces the transfer loss during the mRNA extraction process.

[0090] Kits for constructing mRNA sequencing libraries This disclosure provides a kit for implementing the above-described mRNA sequencing library construction method. The kit contains key reagents or all reagents for implementing all steps of the above-described mRNA sequencing library construction method. The key reagents or all reagents may optionally be packaged separately or in combination in the kit.

[0091] In some specific embodiments, the kit contains an RNase inhibitor and Triton X-100, which are packaged separately or in combination in the kit.

[0092] In some specific embodiments, the RNase inhibitor and Triton X-100 are packaged together as a cell lysis buffer, the cell lysis buffer containing 20-60 U / μL of RNase inhibitor and 5%-12% (w / v) of Triton X-100.

[0093] In some specific embodiments, the kit contains an RNase inhibitor and Triton X-100, which are separately packaged in the kit.

[0094] In some specific embodiments, the separately packaged RNase inhibitor and Triton X-100 are each independently a dry powder formulation or a liquid formulation, and the RNase inhibitor and Triton X-100 can be mixed to prepare a cell lysate with or without the addition of water.

[0095] In some specific embodiments, the cell lysate contains 20-60 U / μL of RNase inhibitor and 5%-12% (w / v) of Triton X-100.

[0096] In some specific embodiments, the cell lysis buffer of this disclosure contains 20-60 U / μL of RNase inhibitor as an RNA protectant; the concentration may be 30-50 U / μL, including but not limited to 20 U / μL, 25 U / μL, 30 U / μL, 35 U / μL, 40 U / μL, 45 U / μL, 50 U / μL, 55 U / μL or 60 U / μL.

[0097] In some specific embodiments, the cell lysis buffer described in this disclosure contains 5% to 12% (w / v) Triton X-100 as a lysis agent, which may be selected as 8% to 12% (w / v), including but not limited to 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), 10% (w / v), 11% (w / v), or 12% (w / v). In some specific embodiments, the cell lysis buffer described in this disclosure consists of 20-60 U / μL of an RNase inhibitor and 10% (w / v) of Triton X-100.

[0098] In some specific embodiments, the cell lysis buffer described in this disclosure consists of 40 U / μL of an RNase inhibitor and 10% (w / v) of Triton X-100.

[0099] In some specific embodiments, the kit may also include other reagents or components that are actually used by those skilled in the art in the process of constructing mRNA sequencing libraries, and the reagents or components may optionally be packaged separately or in combination.

[0100] III. Examples The present disclosure is further illustrated below with reference to embodiments. The description of specific exemplary embodiments of the present disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit the present disclosure to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the teachings of this specification. The exemplary embodiments were chosen and described in order to explain the specific principles of the present disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the present disclosure, as well as various different choices and variations.

[0101] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0102] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0103] Example 1: The method for constructing mRNA sequencing libraries provided in this disclosure 1.1 Preparation of cell lysis buffer Prepare 20 μL of cell lysis buffer with the following composition: 40 U / μL RNase Inhibitor and 10% Triton X-100, using nanofiltered water as the solvent.

[0104] 1.2 Cell lysis Approximately 150,000 human lymphoblasts (Nanjing Kebai Biotechnology Co., Ltd., CBP61543) were placed in cell lysis buffer, centrifuged for 10 seconds, and vortexed for 30 seconds. After vortexing, the cells were briefly centrifuged for 3 seconds and immediately placed in a PCR amplification instrument for incubation at 72°C for 3 minutes. The reaction was then complete.

[0105] 1.3 mRNA enrichment: (1) Prepare the mRNA Capture Beads, Beads Wash Buffer, Tris Buffer, and BeadsBinding Buffer (VAHTS) in advance ® Remove the Universal V10 RNA-seq Library Prep Kit (NR616) from 2-8°C and allow it to equilibrate to room temperature. (2) Add 20 μL of an equal volume of mRNA Capture Beads to the cell lysis product obtained in step 1.2, mix thoroughly, and perform magnetic bead binding reaction in a PCR instrument (65℃, 5 min; 25℃, 5 min). Then elute the mRNA on a magnetic rack; 1.4 RNA Library Construction Use mRNA product kit (VAHTS) ® The Universal V10 RNA-seq Library Prep Kit (NR616) was used to enrich the mRNA of the product from the previous step according to the standard procedure in the product instructions, obtaining mRNA that meets the requirements of Illumina. ® and BGI ® mRNA transcriptome libraries from high-throughput sequencing platforms.

[0106] (1) mRNA fragmentation: Frag / Prime Buffer (1×) is used to fragment the target mRNA. The fragmentation conditions are selected according to the required fragment size.

[0107] (2) First-strand cDNA synthesis: Prepare the reaction system in advance and carry out the first-strand cDNA synthesis reaction in a PCR instrument: heat cover 105℃, 25℃, 10min; 42℃, 15min; 70℃, 15min; 4℃, Hold.

[0108] (3) Second-strand cDNA synthesis: Prepare the second-strand cDNA synthesis reaction system and carry out the second-strand cDNA synthesis reaction in a PCR instrument: heat cover 105℃, 16℃, 30min; 65℃, 15min; 4℃, Hold.

[0109] (4) Adapter ligation and end treatment: Prepare the treatment reagent in advance according to the kit instructions. The reagent contains ligation adapter, end repair reagent and adenine A. Add the cDNA product from the previous step to the reagent in this step and react in the PCR instrument: heat cover 105℃, 20℃, 15min; 4℃, Hold.

[0110] (5) Purification and sorting: Equilibrate the magnetic beads at room temperature in advance, and add 9 μL of magnetic beads to the ligation product. Incubate at room temperature for 10 min to allow DNA to bind to the magnetic beads. Place the sample on a magnetic rack and wait for the solution to become clear. Then, transfer 50 μL of the supernatant to a new PCR tube.

[0111] (6) Washing: Mix the product with purified magnetic beads and incubate at room temperature for 10 min to allow DNA to bind to the magnetic beads; place the sample on a magnetic rack and carefully remove the supernatant after the solution becomes clear; add 100 μL of 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 sec, and carefully remove the supernatant. This step requires two operations.

[0112] (7) Library amplification: Prepare the PCR reaction system and perform a PCR test at 105°C. 98°C, 45 sec. 14 cycles: 98°C, 15 sec; 60°C, 30 sec; 72°C, 30 sec. 72°C, 60 sec; 4°C, hold.

[0113] (8) Product purification: The above library product was purified twice with 0.9× purification magnetic beads to obtain the final library.

[0114] (9) Evaluating the quality of the library Figure 1 The Qsep400 was used to detect the size distribution of DNA fragments in the final library. The fragments were mainly distributed between 200-500 bp, which is a normal distribution of library fragments.

[0115] (10) Sequencing Sequencing: The library was sequenced using the Illumina PE150 platform. After obtaining the fq data, the raw data was first assessed for quality using FastQC software to check the quality status of the raw data. The quality assessment indicators included Q20, Q30, GC content, etc. Next, the raw data was filtered. The filtering conditions were: (1) removing adapter sequences; (2) filtering out sequences containing too many N bases; (3) filtering out sequences with a length of less than 37 bp; (4) filtering out sequences with too many low-quality bases. The filtered data was assessed for quality again, and the samples that passed the quality assessment were analyzed subsequently.

[0116] The parameters used for quality assessment include at least one of the following: (i) Input (number of input reads): refers to the total number of sequencing reads used for alignment analysis. It is usually the number of clean reads obtained after removing adapter sequences and low-quality reads from the raw sequencing data. It is used to characterize the amount of usable sequencing data obtained from the sample.

[0117] (ii) Mapped (number of aligned reads): refers to the number of reads in the above Input reads that can be successfully aligned to the reference genome or reference transcriptome, used to characterize the number of reads with clear genomic location.

[0118] (iii) Map Rate: This refers to the percentage of Mapped reads out of Input reads, i.e., Map Rate = Mapped / Input × 100%. It is used to characterize the purity of the library and the degree of matching between the sequencing reads and the target species. The higher the value, the more reads are derived from the target genome and the better the library quality.

[0119] (iv) FPKM ≥ 0.1 (Effective Gene Expression Index): FPKM (Fragments Per Kilobase of transcript per Million mapped reads) is a unit of measurement for gene expression levels after dual normalization based on gene length and sequencing depth. FPKM ≥ 0.1 refers to genes whose expression levels reach or exceed the 0.1 threshold; levels below this threshold are generally considered unexpressed or background noise. This index represents the number of genes that meet the FPKM ≥ 0.1 standard, indicating the number of effectively detected expressed genes in the sample. A higher value indicates more detected genes.

[0120] (v) rRNA content (ribosomal RNA ratio): This refers to the proportion of reads (or bases) derived from ribosomal RNA (rRNA) in the alignment results. Since rRNA is not the target nucleic acid for transcriptome sequencing, it should be reduced during library construction through mRNA enrichment or rRNA removal; the lower the value, the better the mRNA enrichment effect and the higher the library quality, ideally approaching zero.

[0121] Comparative Example 1: mRNA sequencing library construction 1.1 RNA extraction (1) Sample lysis The cells used in this control example were the same as in Example 1. Cells were collected by centrifugation, and the supernatant was discarded. 500 μL of TRIzol™ Reagent (Thermo Fisher Scientific) was added to each cell tube to a final volume of 1 mL. ®(15596026) Vortex oscillate or repeatedly blow until fully pyrolyzed, then let stand at room temperature for 5 minutes.

[0122] (2) Phase separation (chloroform extraction) Add 0.2 mL of chloroform (Merck) to every 1 mL of TRIzol™ Reagent. ® Shake the solution (1.02445.1000) for 15 seconds until it turns milky pink. Let it stand at room temperature for 2-3 minutes, then centrifuge at 12,000 × g for 15 minutes at 4°C. After centrifugation, aspirate the upper layer (colorless aqueous phase) containing RNA. (3) RNA precipitation Transfer the upper aqueous phase to a new sterile centrifuge tube and add an equal volume of isopropanol (Maclean's). ® Mix well (I811921), let stand at room temperature for 10 minutes, centrifuge at 12,000×g for 10 minutes at 4°C, and a white / transparent gel-like RNA precipitate will be obtained at the bottom of the tube.

[0123] (4) RNA washing Discard the supernatant, add 1 mL of pre-cooled 75% ethanol and gently invert to wash, then centrifuge at 4°C, 7,500 × g for 5 minutes.

[0124] (5) Drying and dissolving Discard the supernatant, remove as much residual ethanol as possible, air dry at room temperature for 5-10 minutes, add 20-50 μL of sterile water, and dissolve by tapping the tube wall or blowing.

[0125] 1.2 RNA Library Construction Use mRNA product kit (VAHTS) ® The Universal V10 RNA-seq Library Prep Kit (NR616) was used to enrich the mRNA of the product from the previous step according to the standard procedure in the product instructions, obtaining mRNA that meets the requirements of Illumina. ® and BGI ® mRNA transcriptome libraries from high-throughput sequencing platforms.

[0126] (1) mRNA enrichment: Add an equal volume of mRNA Capture Beads to the extracted RNA, mix thoroughly, and perform magnetic bead binding reaction in a PCR instrument (65℃, 5 min; 25℃, 5 min). Then elute the mRNA on a magnetic rack; (2) mRNA fragmentation: Frag / Prime Buffer (1×) is used to fragment the target mRNA. The fragmentation conditions are selected according to the required size of the inserted fragment.

[0127] (3) First-strand cDNA synthesis: Prepare the reaction system in advance and carry out the first-strand cDNA synthesis reaction in the PCR instrument: heat cover 105℃, 25℃, 10min; 42℃, 15min; 70℃, 15min; 4℃, Hold.

[0128] (4) Second-strand cDNA synthesis: Prepare the second-strand cDNA synthesis reaction system and carry out the first-strand cDNA synthesis reaction in a PCR instrument: heat cover 105℃, 16℃, 30min; 65℃, 15min; 4℃, Hold.

[0129] (5) Adapter ligation and end treatment: Prepare the treatment reagent in advance according to the kit instructions. The reagent contains ligation adapter, end repair reagent and adenine A. Add the cDNA product from the previous step to the reagent in this step and react in the PCR instrument: heat cover 105℃, 20℃, 15min; 4℃, Hold.

[0130] (6) Purification and sorting: Equilibrate the magnetic beads at room temperature in advance, and add 9 μL of magnetic beads to the ligation product. Incubate at room temperature for 10 min to allow DNA to bind to the magnetic beads. Place the sample on a magnetic rack and wait for the solution to become clear. Then, transfer 50 μL of the supernatant to a new PCR tube.

[0131] (7) Washing: Mix the product with purified magnetic beads and incubate at room temperature for 10 min to allow DNA to bind to the magnetic beads; place the sample on a magnetic rack and carefully remove the supernatant after the solution has clarified; add 100 μL of 80% ethanol (freshly prepared) to rinse the magnetic beads, incubate at room temperature for 30 sec, and carefully remove the supernatant. This step requires two operations.

[0132] (8) Library amplification: Prepare the PCR reaction system and perform a PCR test at 105°C. 98°C, 45 sec. 14 cycles: 98°C, 15 sec; 60°C, 30 sec; 72°C, 30 sec. 72°C, 60 sec; 4°C, hold.

[0133] (9) Product purification: The above library product was purified twice with 0.9× purification magnetic beads to obtain the final library.

[0134] (10) Evaluating the quality of the library Figure 2 The Qsep400 was used to detect the size distribution of DNA fragments in the final library. The fragments were mainly distributed between 200-500 bp, which is a normal distribution of library fragments.

[0135] (11) Sequencing Sequencing: The library was sequenced using the Illumina PE150 platform. After obtaining the fq data, the raw data was first assessed for quality using FastQC software to check the quality status of the raw data. The quality assessment indicators included Q20, Q30, GC content, etc. Next, the raw data was filtered. The filtering conditions were: (1) removing adapter sequences; (2) filtering out sequences containing too many N bases; (3) filtering out sequences with a length of less than 37 bp; (4) filtering out sequences with too many low-quality bases. The filtered data was assessed for quality again, and the samples that passed the quality assessment were analyzed subsequently.

[0136] Example 2: mRNA sequencing library construction 2.1 Cell lysis The cells used in this control example are the same as those in Example 1. 20 μL of lysis buffer (40 U / μL RNase inhibitor and 10% Triton X-100) was added to each cell, and the vortex mixer was set to maximum speed to ensure sufficient cell lysis and vortexed.

[0137] 2.2 Reverse transcription reaction Pre-prepared RT MIX reagent (Picelli, S., Faridani, O., Björklund, Å. et al. F μL l-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171-181(2014). https: / / doi.org / 10.1038 / nprot.2014.006, page 175, Table 1) was aliquoted into PCR tubes. 4.6 μL of the lysed sample was added to the RT MIX, mixed, centrifuged, and incubated in a PCR amplification instrument: 50℃ for 40 min; 90℃ for 1 min; 4℃, holding.

[0138] 2.3 PCR Amplification Prepare the PCR reaction reagent in advance (Picelli, S., Faridani, O., Björklund, Å. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171-181(2014). https: / / doi.org / 10.1038 / nprot.2014.006, p. 175, Table 3). Thaw the reagent on ice, mix well, centrifuge, and prepare the PCR reaction reagent (15 μL). Keep on ice for later use. Add the PCR reaction reagent to each sample tube, mix well, and centrifuge. Perform the PCR program: 95℃ for 3 min, pre-amplification 4 cycles of: 98℃ for 20 s, 65℃ for 30 s, 72℃ for 5 min; then amplify 12 cycles of: 98℃ for 20 s, 67℃ for 15 s, 72℃ for 5 min; 72℃ for 5 min; 4℃, holding. After the PCR amplification reaction was completed, the concentration was measured using a qq.q. meter.

[0139] 2.4 Sample purification cDNA fragments were screened and purified using 0.8×AMP magnetic beads to remove small primer fragments, primer dimers, impurities, etc.

[0140] 2.5 Library Construction The purified cDNA was used to construct a library using the Tn5 transposase method. The Qsep400 quality control library peak chromatogram is shown below. Figure 3 As shown, sequencing was performed.

[0141] 2.6 Sequencing The library was sequenced using the Illumina PE150 platform. After obtaining the fq data, the raw data was first assessed for quality using FastQC software to check its quality. The quality assessment indicators included Q20, Q30, and GC content. Next, the raw data was filtered under the following conditions: (1) removing polya and TSO sequences; (2) removing adapter sequences; (3) filtering out sequences containing too many N bases; (4) filtering out sequences shorter than 37 bp; and (5) filtering out sequences with too many low-quality bases. The filtered data was then assessed for quality again, and the samples that passed the quality assessment were analyzed subsequently.

[0142] Example 2: Comparison of Different Database Construction Methods Using the same cells as in Example 1, two groups were formed based on the number of cells: 2W and 10W. Each group was constructed using the methods provided in Example 1, Control Example 1, and Control Example 2. Two parallel experiments were performed in the 2W group, and three parallel experiments were performed in the 10W group. The quality control results of the constructed mRNA sequencing libraries are shown in Table 1. The genome alignment rate (Map Rate) of the mRNA sequencing library constructed using the method in Example 1 was higher than that of the other two methods. Furthermore, the total number of detectable effective expression genes was the highest at a gene expression value ≥0.1, nearly 20,000. In contrast, the number of detectable effective expression genes in the mRNA sequencing library constructed using the method in Control Example 1 was around 12,000, and the number of detectable effective expression genes in the mRNA sequencing library constructed using the method in Control Example 2 was around 15,000 (see Table 1). Figure 4 In Example 1, the percentage of residual ribosomal RNA (rRNA) was also the lowest (Table 1). Figure 5 The results show that the mRNA sequencing library obtained by the construction method provided in Example 1 has better Pearson correlation than the other two methods.

[0143] Table 1. Data quality comparison of three different methods Verification Example 1 The purpose of this validation example is to further expand the range of cell numbers in cell samples based on the quality detection results of the sequencing libraries obtained by the above-mentioned Example 1, Comparative Example 1 and Comparative Example 2, and to compare the quality detection results of the sequencing libraries obtained by the three construction methods.

[0144] The specific experimental steps were the same as those in Example 1, Control Example 1 and Control Example 2, except that the number of live cells in the cell samples was changed to 1000 groups (1000 live cells), 5000 groups (5000 live cells), 80000 groups (80000 live cells) and 150000 groups (150000 live cells). Each group was tested in three parallel experiments using the three methods described above. The quality test results of the obtained sequencing libraries are shown in the table below.

[0145] Table 2 Comparison of data quality for three different methods As shown in the table above, the Map Rate of the mRNA sequencing library obtained by the construction method provided in Example 1 can reach over 81.5% in the range of 1000 to 150000 live cells, while in Control Examples 1 and 2, the Map Rate is approximately below 80% when the number of live cells is below 5000. The number of effectively expressed genes in the mRNA sequencing library obtained by the construction method provided in Example 1 is not significantly affected by the number of live cells in the sample, maintaining around 20000W in the range of 1000 to 150000, and even under the condition of 1000 to 5000 live cells, the number of effectively expressed genes is still above 19000. In contrast, the number of effectively expressed genes in Control Examples 1 and 2 fluctuates significantly with the number of live cells. Regarding rRNA content, only 2.7% to 3.9% of the rRNA in the mRNA sequencing library obtained by the construction method provided in Example 1 is detected in the range of 1000 to 150000 cells, which is significantly lower than that in Control Examples 1 and 2. In Control Example 2, the number of effectively expressed genes did not show a significant decreasing trend with the increase of cell number. This is because Control Example 2 used a library construction method of direct reverse transcription of complete mRNA, which amplified the difference in the content of high-abundance and low-abundance fragments in the original sample. As the number of cells increased, the difference between the two was further amplified, causing the low-abundance fragments to be diluted and lost after quality control.

[0146] The average effective gene expression levels obtained from 12 experiments using the three library construction methods described above were calculated and compared. Figure 6 As shown, the library construction method provided in Example 1 yielded significantly higher effective gene expression levels in the range of 1000 to 150000 live cells compared to the other two methods. Furthermore, the method provided in Control Example 1 exhibited high dispersion in effective gene expression levels, indicating that its library construction quality was highly dependent on the number of live cells. The correlation of expressed genes in 12 sets of experiments using the three library construction methods was calculated, as shown below. Figure 7 As shown, the mRNA sequencing library obtained by the construction method provided in Example 1 has better Pearson correlation than the other two methods.

[0147] While specific embodiments of this disclosure have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of this disclosure. Therefore, the scope of protection of this disclosure is defined by the appended claims.

Claims

1. A method for constructing an mRNA sequencing library, characterized in that, The construction method includes: Cell samples containing mRNA were lysed using cell lysis buffer, and mRNA was extracted from the cell lysis products by incubation at 20–75°C using an mRNA trapping agent. The mRNA was obtained by elution and fragmented. The fragmented mRNA was used as a template to synthesize the first-strand cDNA, and the first-strand cDNA was used as a template to synthesize the second-strand cDNA to obtain double-stranded cDNA. The double-stranded cDNA was then subjected to end repair and A-tailing, followed by ligation of sequencing adapters and PCR amplification to obtain a sequencing library. The cell lysis buffer consisted of 20-60 U / μL of RNase inhibitor and 10% w / v of Triton X-100; The cell sample contains 1,000 to 150,000 live cells.

2. The construction method according to claim 1, characterized in that, The cell lysis buffer consisted of 40 U / μL of an RNase inhibitor and 10% w / v of Triton X-100.

3. The construction method according to claim 1, characterized in that, The incubation temperature was 72°C.

4. The construction method according to claim 1, characterized in that, The mRNA capturing agent is a magnetic bead bound with oligodeoxythymidine nucleotides.

5. The construction method according to claim 1, characterized in that, The cell sample contains 1,000 to 5,000 live cells.

6. The construction method according to any one of claims 1 to 5, characterized in that, The construction method includes: Cell samples containing mRNA were lysed using cell lysis buffer, and mRNA was extracted from the cell lysis products by incubation at 72°C using magnetic beads bound to oligodeoxythymidine nucleotides. The mRNA was obtained by elution and fragmented. The fragmented mRNA was used as a template to synthesize the first-strand cDNA, and the first-strand cDNA was used as a template to synthesize the second-strand cDNA to obtain double-stranded cDNA. The double-stranded cDNA was then subjected to end repair and A-tailing, followed by ligation of sequencing adapters and PCR amplification to obtain a sequencing library. The cell lysis buffer consisted of 40 U / μL of an RNase inhibitor and 10% w / v of Triton X-100; The cell sample contains 1,000 to 5,000 live cells.

7. A kit for constructing mRNA sequencing libraries, characterized in that, The kit includes a cell lysis buffer consisting of 20–60 U / μL of an RNase inhibitor and 10% w / v of Triton X-100.

8. The reagent kit according to claim 7, characterized in that, The cell lysis buffer consisted of 40 U / μL of an RNase inhibitor and 10% w / v of Triton X-100.

9. A method for detecting mRNA content in cell samples, characterized in that, The detection method includes constructing an mRNA sequencing library of the cell sample using the construction method according to any one of claims 1 to 6, and quantitatively detecting the mRNA content in the mRNA sequencing library.

10. A method for transcriptome sequencing of cell samples, characterized in that, The transcriptome sequencing method uses the construction method described in any one of claims 1 to 6 or the kit described in claim 7 or 8 to construct an mRNA sequencing library and then sequence it.

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