Search

CN-122012676-A - Construction test method, kit and application of single-tube RNAseq sequencing library

CN122012676ACN 122012676 ACN122012676 ACN 122012676ACN-122012676-A

Abstract

The invention discloses a construction method, a kit and an application of a single-tube RNAseq sequencing library, wherein the construction method comprises the following steps of (1) 1 st cDNA synthesis, namely, taking a processed RNA sample, using reverse transcriptase, RNAase Inhibitor, OTR-6N primers and OTR-TSO primers inserted with base U to synthesize first-strand cDNA, (2) library PCR, namely, adding UDG, index PCR PRIMER and 2 XPCR Mix into a reverse transcription product, degrading U base on the OTR-TSO primers by utilizing UDG, directly performing Index PCR amplification, and purifying to obtain the RNAseq sequencing library. The invention forms an RNAseq library construction technology of a cDNA-index PCR amplification-purification' technical route based on the principle that the TdT enzyme activity of reverse transcriptase adds a base at the end of the first strand of cDNA, and forms a kit, and the technology has the advantages of simple operation, reduction of cross contamination between samples and high efficiency and application value.

Inventors

  • ZHU HANYU
  • HUANG KEYU
  • YIN LINGDI
  • LI ZHENYU

Assignees

  • 南京医科大学

Dates

Publication Date
20260512
Application Date
20260408

Claims (10)

  1. 1. A method for constructing a single tube RNAseq sequencing library, comprising the steps of: (1) 1 st cDNA synthesis, namely taking a treated RNA sample, and synthesizing first strand cDNA by using reverse transcriptase, RNAase Inhibitor, OTR-6N primer and OTR-TSO primer inserted with a base U; (2) And library PCR, namely adding UDG, index PCR PRIMER and 2 XPCR Mix into the reverse transcription product, degrading U base on the OTR-TSO primer by using the UDG, directly performing Index PCR amplification, and purifying to obtain the RNAseq sequencing library.
  2. 2. The method for constructing a single tube RNAseq sequencing library according to claim 1, wherein in the step (1), the treated RNA sample is an extracted and purified RNA sample, if the sample is of a complete quality, the sample is subjected to fragmentation treatment, and then the step is performed, and if the sample is severely degraded, the step is directly performed without interruption.
  3. 3. The method of claim 1, wherein in step (1), the OTR-TSO primer is inserted into a base U, wherein the base U is a deoxydu or a non-deoxyu.
  4. 4. The method for constructing a single tube RNAseq sequencing library according to claim 1, wherein in the step (1), the nucleotide sequence of the OTR-6N primer is shown as SEQ ID NO.1, and the nucleotide sequence of the OTR-TSO primer inserted into the base U is shown as SEQ ID NO. 2.
  5. 5. The method for constructing a single tube RNAseq sequencing library according to claim 1, wherein in step (2), index PCR PRIMER comprises a Primer i5-Primer and a Primer i7-Primer, the nucleotide sequence of the Primer i5-Primer is shown as SEQ ID NO.3, and the nucleotide sequence of the Primer i7-Primer is shown as SEQ ID NO. 4.
  6. 6. A kit for single tube RNAseq sequencing library construction, comprising OTR-TSO primers inserted into base U.
  7. 7. The kit according to claim 6, wherein the OTR-TSO primer inserted into the base U has a nucleotide sequence shown in SEQ ID NO. 2.
  8. 8. The kit of claim 6, wherein the kit comprises: RNA fragmentation reagent, reverse transcriptase, OTR-6N primer, OTR-TSO primer inserted into base U, index PCR PRIMER and uracil-DNA glycosidase UDG.
  9. 9. The kit according to claim 8, wherein the OTR-6N Primer has a nucleotide sequence shown in SEQ ID NO.1, the Index PCR PRIMER comprises primers i5-Primer and i7-Primer, the nucleotide sequence of the i5-Primer is shown in SEQ ID NO.3, and the nucleotide sequence of the i7-Primer is shown in SEQ ID NO. 4.
  10. 10. Use of a kit according to any one of claims 6 to 9 in the construction of a single tube RNAseq sequencing library.

Description

Construction test method, kit and application of single-tube RNAseq sequencing library Technical Field The invention belongs to the technical field of RNAseq sequencing libraries, and particularly relates to a construction test method, a kit and application of a single-tube RNAseq sequencing library. Background The method for constructing the RNAseq high-throughput sequencing library is subjected to a plurality of iterations and updates. Starting from the original technical route of cDNA single-strand synthesis-cDNA double-strand synthesis-cDNA purification-cDNA disruption and end repair-purification-3 ' end addition A-product purification-adaptor-ligation product purification-index PCR amplification-library purification ', a relatively efficient technical route of cDNA single-strand synthesis-cDNA double-strand synthesis and end repair and 3' end addition A-adaptor ligation product-purification-index PCR amplification-library purification is formed through continuous efforts of various large enterprises worldwide, so that experimental procedures are greatly simplified, experimental time is saved, and experimental efficiency is improved. With the intensive research and accumulation of application experience on the activity of reverse transcribed terminal transferase (TdT), a simplified technical route of 'cDNA one-strand synthesis-first strand cDNA purification-index PCR amplification-library purification' was developed. Although the above-mentioned technique is very simple, the whole experimental procedure consists of multiple tube-turning experimental operations, and especially before index PCR amplification, the purification steps are prone to error and cause cross contamination between samples, thereby affecting the experimental results. Therefore, the construction of the RNAseq high-throughput sequencing library can reduce experimental operation as much as possible and reduce the number of times of tube transfer before index PCR amplification, thereby being beneficial to the quality of the RNAseq library and improving experimental results. Disclosure of Invention Aiming at the problems in the prior art, the invention provides a construction test method, a kit and application of a single-tube RNAseq sequencing library. The RNAseq library construction technology is a single tube mode of 'cDNA-index PCR amplification-purification by RNA reverse transcription', and the experimental flow from cDNA synthesis to index PCR amplification is carried out in the same PCR tube, so that an effective RNAseq library is finally formed. The technical scheme adopted by the invention is as follows in order to achieve the aim of the invention: in a first aspect, the invention provides a method for constructing a single tube RNAseq sequencing library, comprising the steps of: (1) 1 st cDNA synthesis, namely taking a treated RNA sample, and synthesizing first strand cDNA by using reverse transcriptase, RNAase Inhibitor, OTR-6N primer and OTR-TSO primer inserted with a base U; (2) And library PCR, namely adding UDG, index PCR PRIMER and 2 XPCR Mix into the reverse transcription product, degrading U base on the OTR-TSO primer by using the UDG, directly performing Index PCR amplification, and purifying to obtain the RNAseq sequencing library. In the step (1), the treated RNA sample refers to the RNA sample after extraction and purification, if the sample is complete in quality, the sample needs to be fragmented first and then treated in the step, and if the sample is severely degraded, the sample does not need to be broken, and the treatment in the step is directly carried out. As a specific embodiment, in step (1), the OTR-TSO primer is inserted with a base U, wherein the base U is a deoxidized dU or a non-deoxidized U. In the step (1), the nucleotide sequence of the OTR-6N primer is shown as SEQ ID NO.1, and the nucleotide sequence of the OTR-TSO primer inserted with the base U is shown as SEQ ID NO. 2. As a specific embodiment, in the step (2), the Index PCR PRIMER comprises a Primer i5-Primer and a Primer i7-Primer, the nucleotide sequence of the Primer i5-Primer is shown as SEQ ID NO.3, and the nucleotide sequence of the Primer i7-Primer is shown as SEQ ID NO. 4. In a second aspect, the invention provides a kit for single tube RNAseq sequencing library construction, the kit comprising an OTR-TSO primer inserted into base U. As a specific embodiment, the nucleotide sequence of the OTR-TSO primer inserted into the base U is shown as SEQ ID NO. 2. As a specific embodiment, the kit comprises: RNA fragmentation reagent, reverse transcriptase, OTR-6N primer, OTR-TSO primer inserted into base U, index PCR PRIMER and uracil-DNA glycosidase UDG. As a further scheme, the nucleotide sequence of the OTR-6N Primer is shown as SEQ ID NO.1, the Index PCR PRIMER comprises a Primer i5-Primer and a Primer i7-Primer, the nucleotide sequence of the i5-Primer is shown as SEQ ID NO.3, and the nucleotide sequence of the i7-Primer is shown as SEQ