Reverse transcription system, pre-amplification system, enzyme digestion interruption system and application

Abstract

The invention relates to the field of biological sequencing, in particular to a reverse transcription system, a pre-amplification system, an enzyme digestion interruption system and application. The invention provides a reverse transcription system which comprises a reverse transcription buffer solution, a reverse transcription primer and reverse transcriptase, the reverse transcription buffer solution is prepared from the following components: 5XFirstStandBuffer, 5MBeta and 100mM of DTT (Dimethyl Terephthalate); the reverse transcription primer comprises 100mM < MgCl2 > and 100mu m < MTSO >; the reverse transcriptase comprises the following components: 40 U / [mu] L of RNA (Ribonucleic Acid) sensor and 200 U / [mu] L of Alpha ReverseTransscriptase. Based on the basic principle of Smart-seq and the prior art, the system and the existing formula of the Smart-seq experimental process are adjusted, so that the full-length transcript of the single cell is successfully amplified, and certain stability is kept.

Description

Technical Field

[0001] The present invention relates to the field of biological sequencing, and in particular to a reverse transcription system, a pre-amplification system, an enzyme cutting and interruption system and applications thereof. Background Art

[0002] Smart-seq is a single-cell full-length RNA-seq method with good coverage uniformity and detection sensitivity, and is widely used in single-cell transcriptome analysis. The full name of the SMART method is Switching Mechanism at 5'End of RNA Template. The reason why the full-length amplification of the transcriptome can be achieved is mainly based on two points: first, a positioning primer is used to ensure that the synthesis of cDNA starts from the 3' end of the mRNA, and at the same time, the synthesized cDNA is connected to a universal PCR sequence downstream; second, the MMLV reverse transcriptase will add a few more C bases to the 3' end of the newly synthesized cDNA, and then use an upstream primer with 3 G bases to synthesize the second chain. This ensures that only the complete first-chain cDNA, that is, those cDNAs with multiple "C" (first chain), can be synthesized into the second chain of cDNA, which also ensures that the double-stranded cDNA is full-length cDNA. Because complete transcriptome information can be obtained, Smart-seq plays an important role in single-cell transcriptome analysis.

[0003] The principle of fragmentase library construction is to use fragmentase to randomly interrupt the target sequence (cDNA), then perform end repair, use rTaq enzyme to add A at the end, then connect Ad153_2B adapter, purify the adapter ligation product, and finally use primers to PCR amplify the fragmented cDNA, purify the fragments, circularize, make DNB and sequence.

[0004] Conventional Smart-seq library construction technology is to conduct reactions in 384-well plates, use flow cytometry to sort single cells for reverse transcription to obtain full-length transcriptomes and amplify cDNA, and then use fragmentation enzymes to perform subsequent fragmentation experiments on cDNA for library construction. However, since two sequences are embedded in Tn5, only products that embed these two sequences at the same time can be amplified, so only one-third of the sequences can be obtained, so the library construction effect is not good, and the reverse transcription effect is not good.

[0005] Therefore, there is an urgent need for a library construction technology with better library construction effect and better reverse transcription effect. Summary of the invention

[0006] In view of this, the present invention provides a reverse transcription system, a pre-amplification system, an enzyme cleavage interruption system and applications. Based on the basic principles of Smart-seq and existing technologies, the present invention adjusts the system and existing formula of the Smart-seq experimental process to achieve the purpose of successfully amplifying the full-length transcript of a single cell and maintaining a certain stability.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a reverse transcription system, comprising: a reverse transcription buffer, a reverse transcription primer and a reverse transcriptase;

[0009] The reverse transcription buffer comprises:

[0010] First Strand Buffer 5X

[0011] Betaine 5M

[0012] DTT 100 mM;

[0013] The reverse transcription primer comprises:

[0014] MgCl2 100mM

[0015] TSO 100 μM;

[0016] The reverse transcriptase comprises:

[0017] RNAse inhibitor 40U / μL

[0018] Alpha Reverse Transcriptase 200U / μL.

[0019] In some embodiments of the present invention, the reverse transcription system comprises:

[0020] Reverse transcription buffer 0.75 μL

[0021] Reverse transcription primer 0.25 μL

[0022] Reverse transcriptase 0.2 μL.

[0023] In the above transcription system, the reverse transcription buffer comprises:

[0024] 5X First Strand Buffer 0.245μL

[0025] 5M Betaine 0.445μL

[0026] 100mM DTT 0.06μL;

[0027] The reverse transcription primer comprises:

[0028] 100mM MgCl2 0.248μL

[0029] 100 μM TSO 0.002 μL;

[0030] The reverse transcription primer comprises:

[0031] 40U / μL RNAse inhibitor 0.075μL

[0032] 200U / μL Alpha Reverse Transcriptase 0.125μL.

[0033] The present invention also provides a pre-amplification system, comprising:

[0034] GoldenHigh-Fidelity Readymix 2X IS PCR Primer 10 μM.

[0035] In some embodiments of the present invention, the above-mentioned pre-amplification system comprises:

[0036] GoldenHigh-Fidelity Readymix 1.55μL IS PCR Primer 0.05 μL.

[0037] The present invention provides an enzyme cleavage interruption system, comprising:

[0038] Fragmentase buffer 10X Fragmentase 0.05~0.1μL.

[0039] In some embodiments of the present invention, the above-mentioned enzyme cleavage and disruption system comprises:

[0040] 10XFragmentase buffer 0.2μL Fragmentase 0.05~0.1μL NF-H2O 0.1 μL.

[0041] In some embodiments of the present invention, the above-mentioned enzyme cleavage and disruption system comprises:

[0042] 10XFragmentase buffer 0.2μL Fragmentase 0.1μL NF-H2O 0.1 μL.

[0043] The present invention also provides library construction reagents, including: the above-mentioned reverse transcription system, the above-mentioned pre-amplification system and / or the above-mentioned enzyme cleavage interruption system.

[0044] The present invention also provides a method for constructing a library, wherein the sample to be tested is lysed, reverse transcribed, pre-amplified, and cut by enzyme, and then the end is repaired, the adapter is connected, pooling is performed for purification, amplification, screening, and circularization to obtain a library;

[0045] The reverse transcription adopts the reverse transcription system mentioned above or the reverse transcription system in the library construction reagent mentioned above;

[0046] The pre-amplification adopts the pre-amplification system mentioned above or the pre-amplification system in the library construction reagent mentioned above;

[0047] The enzymatic cleavage and disruption adopt the above enzymatic cleavage and disruption system or the enzymatic cleavage and disruption system in the above library construction reagent.

[0048] In some embodiments of the present invention, in the above construction method, the enzyme cleavage interruption procedure includes:

[0049]

[0050] In some embodiments of the present invention, in the above construction method, the enzyme cleavage interruption procedure includes:

[0051]

[0052] In some embodiments of the present invention, in the above construction method, the lysis uses a cell lysis reagent, and the cell lysis reagent includes:

[0053]

[0054] In some embodiments of the present invention, in the above construction method, the cell lysis reagent comprises:

[0055]

[0056] In some embodiments of the present invention, in the above construction method, the cleavage procedure includes:

[0057] Temperature Time 72℃ 5min

[0058] 4℃∞.

[0059] In some embodiments of the present invention, in the above construction method, the reverse transcription procedure includes:

[0060]

[0061] In some embodiments of the present invention, in the above construction method, the pre-amplification procedure includes:

[0062]

[0063] In some embodiments of the present invention, in the above construction method, the repairing uses a repair reagent, and the repair reagent comprises:

[0064]

[0065] In some embodiments of the present invention, in the above construction method, the repair procedure includes:

[0066] 37℃ 30min

[0067] 65℃ 15min

[0068] Cool down at 0.1℃ / s to 4℃∞.

[0069] In some embodiments of the present invention, in the above construction method, the linker connection uses a linker connection reagent, and the linker connection reagent includes:

[0070]

[0071] In some embodiments of the present invention, in the above construction method, the pooling purification uses 0.6XAgencourtAMPureXP beads.

[0072] In some embodiments of the present invention, in the above construction method, the amplification adopts a PCR system, and the PCR system comprises:

[0073]

[0074] The X is 400 divided by the concentration.

[0075] In some embodiments of the present invention, in the above construction method, the amplification procedure includes:

[0076]

[0077] In some embodiments of the present invention, in the above construction method, the screening uses 0.4X and 0.3X Agencourt AMPure XP beads.

[0078] In some embodiments of the present invention, in the above construction method, the cyclization uses a cyclization reagent, and the cyclization reagent includes:

[0079]

[0080] In some embodiments of the present invention, in the above construction method, the cyclization further comprises a step of single-stranded DNA ligation; the single-stranded DNA ligation adopts a single-stranded DNA ligation system, and the single-stranded DNA ligation system comprises:

[0081]

[0082] In some embodiments of the present invention, in the above construction method, the single-stranded DNA connection is carried out by incubating at 37° C. for 1 hour and then maintaining at 4° C.

[0083] In some embodiments of the present invention, in the above construction method, after the cyclization, the steps of exonuclease digestion and magnetic bead purification are also included.

[0084] In some embodiments of the present invention, in the above construction method, the exonuclease digestion uses a digestion reagent, and the digestion reagent includes:

[0085]

[0086] In some embodiments of the present invention, in the above construction method, the exonuclease digestion is performed by incubating at 37° C. for 30 min and then maintaining at 4° C.

[0087] In some embodiments of the present invention, in the above-mentioned construction method, the magnetic bead purification step includes: taking magnetic beads and mixing them with the circularized sample to be tested, incubating, taking the supernatant, rinsing, drying, eluting, and purifying to obtain ssDNA.

[0088] In some embodiments of the present invention, in the above construction method, the volume ratio of the reverse transcription system, the pre-amplification system and the enzyme cleavage and disruption system is 3:4:1.

[0089] The present invention also provides the use of the reverse transcription system, the pre-amplification system, the enzyme cleavage disruption system and / or the library construction reagent in library construction.

[0090] The present invention also provides a kit, comprising the reverse transcription system, the pre-amplification system, the enzyme cleavage and disruption system and / or the library construction reagents and an acceptable auxiliary agent or carrier.

[0091] In some embodiments of the present invention, the above-mentioned kit further comprises: one or more of a primer set, a cell lysis reagent, a repair reagent, a linker ligation reagent, an amplification reagent, a circularization reagent and / or a digestion reagent.

[0092] The present invention also provides a library obtained by the above construction method.

[0093] The present invention also provides application of the library in gene detection.

[0094] The present invention provides a reverse transcription system, comprising: a reverse transcription buffer, a reverse transcription primer and a reverse transcriptase;

[0095] The reverse transcription buffer comprises:

[0096] First Strand Buffer 5X

[0097] Betaine 5M

[0098] DTT 100 mM;

[0099] The reverse transcription primer comprises:

[0100] MgCl2 100mM

[0101] TSO 100 μM;

[0102] The reverse transcriptase comprises:

[0103] RNAse inhibitor 40U / μL

[0104] Alpha Reverse Transcriptase 200U / μL.

[0105] The beneficial effects of the present invention include:

[0106] (1) The present invention changes the reverse transcription (RT) system to improve the reverse transcription efficiency.

[0107] (2) The present invention changes the pre-amplification (pre-PCR) system and solves the problem of reagent costs through trial and error.

[0108] (3) In order to ensure a better disruption effect and cooperate with a small system, the present invention replaces the Tn5 disruption enzyme in the original enzyme cleavage disruption system with Fragmentase, thereby improving the disruption efficiency, and determines the optimal amount of disruption enzyme and disruption time through trial and error.

[0109] (4) The system provided by the present invention is trace and the reagent cost is low.

[0110] (5) The system provided by the present invention can directly build a library for all cDNAs in a single tube, making the experimental operation more convenient.

[0111] (6) The system provided by the present invention can improve the efficiency of cDNA shearing and increase the sample yield.

[0112] (7) The system and construction method provided by the present invention can be used to achieve automated high-throughput using a sample pipetting instrument.

[0113] (8) The system and construction method provided by the present invention ensure the utilization rate of rare samples.

[0114] (9) The construction method provided by the present invention has low dependence on instruments. The experiment can be completed with only a suitable automatic loading workstation, without the need to build an expensive single-cell platform, such as a microfluidic platform.

[0115] (10) The stability of this technology was verified multiple times, and the results showed that the effect was stable. The sequencing results of the BGIseq500 sequencing platform also verified the feasibility of this technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0117] Figure 1 Show the basic experimental process of Smart-seq based on BGISEQ500 platform;

[0118] Figure 2 The figure shows the result of the interruption amplification when the amount of the interruption enzyme is 0.1 μL and the interruption is performed for 5 minutes; wherein: the ordinate is the fluorescence intensity, representing the number of fragments;

[0119] Figure 3 The figure shows the result of the interruption amplification when the amount of the interruption enzyme is 0.05 μL and the interruption is performed for 7 minutes; wherein: the ordinate is the fluorescence intensity, representing the number of fragments;

[0120] Figure 4 The figure shows the result of the interruption amplification when the amount of the interruption enzyme is 0.1 μL and the interruption is performed for 7 minutes; wherein: the ordinate is the fluorescence intensity, representing the number of fragments;

[0121] Figure 5 The results of qPCR product detection are shown, where: 2100 fragment range 500-2000 bp, concentration>3ng / μL;

[0122] Figure 6 The results of the interrupted PCR product detection are shown, where: the 2100 fragment ranges from 300 to 1000 bp and the concentration is >50 ng / μL. DETAILED DESCRIPTION

[0123] The invention discloses a reverse transcription system, a pre-amplification system, an enzyme cutting and interruption system and applications.

[0124] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0125] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0126] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0127] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0128] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0129] The present invention modifies the conventional Smart-seq system, but does not simply reduce or increase the system in proportion to the existing process. We change the fragmentation enzyme in the original system to improve the fragmentation efficiency and successfully construct a library, which can be automated in conjunction with a sample pipetting instrument.

[0130] In Example 1, Effect Example 1 and Effect Example 2 of the present invention, the raw materials and reagents used can be purchased from the market.

[0131]

[0132]

[0133] Primer sequences:

[0134]

[0135]

[0136] The present invention will be further described below in conjunction with embodiments:

[0137] Example 1

[0138] Based on the basic principles of Smart-seq and existing technologies, the system and existing formula of the Smart-seq experimental process are adjusted to achieve the goal of successfully amplifying single-cell full-length transcripts and maintaining a certain stability.

[0139] (1) Prepare lysis solution

[0140] Reagents Volume (μL) RNA Spike-In (1:5,000,000) 0.1 10μM Oligo-dT Primer 0.02 40U / μL RNase Inhibitor 0.0625 10% Triton X-100 0.0125 5X First Strand Buffer 0.25 10mM dNTP 0.45 NF water 0.305 total 1.2

[0141] (2) Cell lysis: Single cells of Hela cells were sorted into the lysis buffer of step (1) and centrifuged at high speed for 10 seconds. Set the PCR program for lysis and heat the lid at 75°C.

[0142] temperature time 72℃ 5min 4℃ ∞

[0143] (3) Reverse transcription: After cell lysis, add the following reverse transcription system and centrifuge to mix the solution thoroughly.

[0144] Reagents Volume (μL) RT Buffer 0.75 RT Primer 0.25 RT Enzyme 0.2 total 1.2

[0145] RT Buffer (reverse transcription buffer): 0.245 μL 5X First Strand Buffer, 0.445 μL 5M Betaine, 0.06 μL 100 mM DTT;

[0146] RT Primer (reverse transcription primer): 0.248 μL 100 mM MgCl2, 0.002 μL 100 μM TSO;

[0147] RT Enzyme (reverse transcriptase): 0.075μL 40U / μL RNAse inhibitor, 0.125μL 200U / μL Alpha Reverse Transcriptase.

[0148] Set up the PCR program according to the table below and start reverse transcription:

[0149]

[0150] (4) Pre-PCR: Add the pre-amplification system and mix the solution thoroughly.

[0151] Reagents Volume (μL) PCR Mix 1.55 10μM IS PCR Primer 0.05 Total 1.6

[0152] PCR Mix (preamplification reaction solution: 2X Golden High-Fidelity Readymix, Thermo;

[0153] IS PCR Primer (pre-amplification primer);

[0154] Set the PCR program according to the table below, with the heated cover at 105°C:

[0155]

[0156] like Figure 5 As shown, this step performs quality control based on the 2100 curve to determine whether a product is generated.

[0157] (5) Enzymatic digestion: Add the enzyme digestion system, gently blow to mix, and centrifuge to fully mix the solution.

[0158] Reagents Volume (μL) 10X fragmentase buffer 0.2 Fragmentase 0.05～0.1 <![CDATA[NF-H2O]]> 0.1 Total 0.4

[0159] Set the PCR program according to the table below, with a heated cover at 75°C:

[0160] temperature time 4℃ 5min 37℃ 5min 65℃ 15min 4℃ ∞

[0161] like Figure 6 As shown, this step performs quality control based on the 2100 curve to determine the fragment range of the interrupted product.

[0162] (6) End repair plus A: Add 0.4 μL of the enzyme cleavage system in step (5), gently pipette to mix, and centrifuge to fully mix the solution.

[0163] Reagents Volume (μL) 10X PNKbuffer 0.52 20:1 dATP:dNTP mix (1.25 mM) 0.14 T4 PNK (10U / μL) 0.06 T4 DNA polymerase (3U / μL) 0.06 rTaq(5U / μL) 0.02 Total 0.8

[0164] Set the PCR program according to the table below, with a heated cover at 75°C:

[0165] temperature time 37℃ 30min 65℃ 15min 0.1℃ / s cooling to 4℃ ∞

[0166] (7) Connector connection: Add the sample obtained in step (6) to the connector connection system, gently blow to mix, and centrifuge to fully mix the solution.

[0167]

[0168]

[0169] Set the PCR program according to the table below, with a heated cover at 75°C:

[0170] temperature time 23℃ 60min 4℃ ∞

[0171] (8) Sample pooling and purification

[0172] All samples obtained in step (7) were taken out and pooled into a library. Each library was purified using 0.6X Agencourt AMPure XP beads;

[0173] In this step, quality control is performed based on the 2100 curve to check the purification effect of the product.

[0174] (9) PCR: Add the sample obtained in step (8) into the reaction system shown in the table below, vortex and centrifuge.

[0175] (X=400 / concentration)

[0176] Reagents Volume (μL) 2X Golden High-Fidelity Readymix 90 Ad153 F primer (10 μM) 7.2 Ad153 R primer (10 μM) 7.2 DNA X <![CDATA[NF-H2O]]> 75.6-X Total 180

[0177] Set the PCR program according to the table below, with the heated cover at 105°C:

[0178]

[0179]

[0180] (10) Pooling purification fragment screening

[0181] Take 5 μL of each sample obtained in step (9) and pool them into a library. Each library is selectively purified using 0.4X and 0.3X Agencourt AMPure XP beads.

[0182] In this step, the double-stranded DNA concentration was detected using Qubit.

[0183] (11) Cyclization

[0184] a) Prepare the reagents according to the following formula: (X = 550 / concentration)

[0185] Reagent Volume(μL) DNA Mix X Ad153 splint oligo (20 μM) 5 Water Make up to 70 μL Total 70

[0186] b) Set the parameters according to the following procedure:

[0187] temperature time 95℃ 3min 12℃ hold

[0188] c) ssDNA ligation, place the DNA Mix tube obtained in b) in an ice bath and add each reaction component in sequence:

[0189] Reagents Volume (μL) DNA Mix 70μL 10X TA buffer 12μL 100mM dATP 1.2μL T4 DNA Ligase (600U / μL) 0.42μL Water 36.38μL Total 120μL

[0190] d) After preparing the above reaction solution, vortex and shake gently for 5 seconds; incubate at 37℃ for 1 hour; and hold at 4℃.

[0191] (12)Exo Digestion

[0192] a) Place the Ligated DNA Mix obtained in step (11) in an ice bath and add each reaction component in sequence:

[0193] Reagents Volume (μL) Ligated DNA Mix 120 10X TA buffer 0.8 Exo I (20U / μL) 3.9 Exo III (100U / μL) 1.3 Water 2 Total 128

[0194] b) After preparing the above reaction solution, vortex and shake gently for 5 seconds; incubate at 37℃ for 30 minutes and hold at 4℃; take 1μL of the product after enzyme digestion to measure the DNA concentration.

[0195] (13) Magnetic bead purification

[0196] a) Take 170 μL of PEG32 beads and add them to the reaction solution obtained in step (12), vortex and mix well, and incubate at room temperature (25°C ± 5°C) for 10 minutes;

[0197] b) Briefly centrifuge the reaction tube and place it on a magnetic stand to separate the beads and liquid. Wait for the solution to become clear (approximately 5 minutes) and carefully remove the supernatant;

[0198] c) Keep the EP tube in the magnetic rack and add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature (25℃±5℃) for 30 seconds and carefully remove the supernatant;

[0199] d) Repeat the above step, rinsing twice in total;

[0200] e) Keep the EP tube in the magnetic rack and open the cover to air-dry the magnetic beads for 10 minutes;

[0201] f) Remove the EP tube from the magnetic rack and add 40 μL sterile ultrapure water for elution. Vortex or use a pipette to gently blow and mix thoroughly. Centrifuge the reaction tube briefly and place it on the magnetic rack to separate the magnetic beads and liquid. When the solution is clear (about 5 minutes), carefully pipette the supernatant into a sterile EP tube and store at -20°C;

[0202] g) Take 1 μL of the purified product to measure the ssDNA concentration; store at -20°C until sequencing.

[0203] Effect Example 1

[0204] like Figure 2 As shown, when the amount of fragmentation enzyme (Fragmentase) in step (5) of Example 1 is 0.1 μL and the fragmentation is performed for 5 minutes, the fragments are too large.

[0205] like Figure 3 As shown, when the amount of the disrupting enzyme in step (5) of Example 1 was reduced to 0.05 μL and the disruption was carried out for 7 minutes, the fragments were too large and the amount of enzyme was insufficient.

[0206] like Figure 4As shown, when the enzyme amount in step (5) of Example 1 was increased to 0.1 μL and the fragments were fragmented for 7 minutes, the fragments were mainly distributed in the range of 200 to 1000 bp, which better met the requirements of subsequent library construction.

[0207] Figure 2 to Figure 4 The ordinate is the fluorescence intensity, which represents the number of fragments. From the peak shape, Figure 4 The number of fragments with the horizontal axis between 200-1000 bp is relatively large. Figure 2 and Figure 3 many.

[0208] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A reverse transcription system, characterized in that: include: Reverse transcription buffer, reverse transcription primers, and reverse transcriptase; The reverse transcription buffer comprises: First StrandBuffer 5XBetaine 5MDTT 100mM; The reverse transcription primer comprises: MgCl2 100mMTSO 100μM; The reverse transcriptase comprises: RNAse inhibitor 40U / μLAlphaReverse Transcriptase 200U / μL.

2. A pre-amplification system, characterized in that: include: GoldenHigh-Fidelity Readymix 2XIS PCRPrimer 10μM.

3. Enzyme cutting interruption system, characterized in that: include: Fragmentase buffer 10X Fragmentase 0.05~0.1μL.

4. Library construction reagent, characterized in that, include: The reverse transcription system according to claim 1, the pre-amplification system according to claim 2 and / or the enzyme cleavage and disruption system according to claim 3.

5. A method for constructing a library, characterized in that: After the sample to be tested is lysed, reverse transcribed, pre-amplified, and cut by enzyme, the end is repaired, the adapter is connected, pooling is performed, amplified, screened, and circularized to obtain the library; The reverse transcription adopts the reverse transcription system as claimed in claim 1 or the reverse transcription system in the library construction reagent as claimed in claim 4; The pre-amplification adopts the pre-amplification system as claimed in claim 2 or the pre-amplification system in the library construction reagent as claimed in claim 4; The enzymatic cleavage and disruption adopts the enzymatic cleavage and disruption system as described in claim 3 or the enzymatic cleavage and disruption system in the library construction reagent as described in claim 4.

6. The construction method according to claim 5, characterized in that: The enzyme cleavage interruption procedure includes:

7. The construction method according to claim 5 or 6, characterized in that: The volume ratio of the reverse transcription system, the pre-amplification system and the enzyme cleavage and disruption system is 3:4:

1.

8. Use of the reverse transcription system according to claim 1, the pre-amplification system according to claim 2, the enzyme cleavage and disruption system according to claim 3 and / or the library construction reagent according to claim 4 in library construction.

9. A kit, characterized in that It comprises the reverse transcription system as claimed in claim 1, the pre-amplification system as claimed in claim 2, the enzyme cleavage and disruption system as claimed in claim 3 and / or the library construction reagent as claimed in claim 4 and an acceptable auxiliary agent or carrier.

10. The kit according to claim 9, characterized in that Also includes: One or more of a primer set, a cell lysis reagent, a repair reagent, an adapter ligation reagent, an amplification reagent, a circularization reagent, and / or a digestion reagent.

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