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RNA library building method

A library and magnetic bead technology, applied in the field of RNA library construction, can solve the problems of small dosage, complex steps, long library construction period, etc., and achieve the effects of simple experimental steps, flexible input, and reduced dependence.

Pending Publication Date: 2021-01-05
上海英基生物科技有限公司
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method takes a long time to build a library, the steps are complicated, and error-prone
[0003] In addition, the amount of a traditional Tn5 transposase is only for the input amount of specific RNA, and the transposase has a strong dependence on the amount of template input.
[0004] The document "RNA sequencing by direct tagmentation of RNA / DNA hybrids" (DiLin, Fu Yusi, Sun Yue, et al. Proc Natl Acad Sci USA. 2020, 117(6): 2886-2893.) discloses a new method based on Compared with other existing methods, the rapid library construction method of transcriptome sequencing of Tn5 transposase greatly simplifies the process of library construction; at the same time, the amount of sample is still very small (10ng-100pg), which can not only be used for high-quality Single-cell transcriptome sequencing, and the sequencing quality and speed of samples such as the new coronavirus (2019-nCoV) may be improved to a certain extent, but it still has certain limitations on the range of sample volume, which affects efficiency when used
[0005] Therefore, it is necessary to study a rapid RNA sequencing library construction method with a wider range of sample consumption. By reducing the dependence of transposase on template input, it overcomes the defects of long library construction cycle, complicated steps, and error-prone. While building a library flexibly, expand the scope of application of sample volume

Method used

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  • RNA library building method
  • RNA library building method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0036] Implementation Case 1: Assembly of Transposome Complex and Magnetic Beads

[0037] Step 1: DNA linker primer preparation

[0038] The DNA linker primer contains mosaic end sequences recognized by transposase and a sequence connected to magnetic beads. The specific sequence is as follows:

[0039] Biotin-Tn5 ME-A: 5'-NNNNNNNNNN(5-100bp)-TCGTC GGCAG CGTCA GATGT GTATAAGAGA CAG-3' 5'-Biotin

[0040] Biotin-Tn5 ME-B: 5'-NNNNNNNNNNNN(5-100bp)-GTCTC GTGGG CTCGG AGATG TGTATAAGAG ACAG-3' 5'-Biotin Tn5 MErev: 5'-CTGTCTCTTATACACATCT-3' 5'-phosphorylation modification

[0041] Step 2: Assembly of the transposase complex

[0042]The DNA linker annealing buffer, transposase assembly buffer, and Tn5 transposase were provided by the One-step DNA Lib prep kit produced by Wuhan Aibtech Biotechnology Co., Ltd.

[0043] 2.1 DNA linker assembly

[0044] Dissolve the DNA linker sequence in 1xTE buffer, adjust the concentration to 100uM, and assemble the DNA linker with Biotin-Tn5ME-A seq...

Embodiment 2

[0053] Example 2 Building a library with different total RNA inputs in the same species

[0054] 1. Using Oligo(dT) 23 VN for first cDNA synthesis

[0055] Start with total RNA input, respectively input 10ng, 100ng, 1ug ​​mouse total RNA to directly synthesize first cDNA.

[0056] 1. first cDNA synthesis

[0057] 1.1 Take out the Total RNA sample, thaw it on ice, dilute 10ng~1ug sample with Nuclease-free Water to 8μL, and keep it on ice for later use;

[0058] 1.2 Take out 5X First Strand Buffer, Oligo(dT) 23 VN was melted on ice, and the reaction system was prepared according to the following table on ice:

[0059]

[0060]

[0061] *First Strand Synthesis Enzyme Mix components include reverse transcriptase and RNA inhibitor, the reverse transcriptase is ABSScript III reverse transcriptase (ABclonal RK20408) or ABSScript II reverse transcriptase (ABclonalRK21400) or other commercially available reverse transcriptases capable of synthesizing full-length first cDNA r...

Embodiment example 3

[0148] Implementation case 3. Total RNA library construction of different species with the same input amount

[0149] 1. Using Oligo(dT) 23 VN for first cDNA synthesis

[0150] Start with total RNA input, and input the same amount of human, rat, and mouse total RNA respectively to directly synthesize first cDNA.

[0151] 1. first cDNA synthesis

[0152] 1.1 Take out the Total RNA sample, thaw it on ice, dilute 10ng~1ug sample with Nuclease-free Water to 8μL, and keep it on ice for later use.

[0153] 1.2 Take out 5X First Strand Buffer, Oligo(dT) 23 VN was melted on ice, and the reaction system was prepared according to the following table on ice:

[0154] Reagent volume Total RNA sample 8μL 5X First Strand Buffer 4μL Betaine 4μL Oligo(dT)23VN 2μL First Strand Synthesis Enzyme Mix 2μL total capacity 20 μL

[0155] 1.3 Use a pipette to mix evenly, centrifuge briefly, and place the system in a PCR instrument (75°C ho...

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Abstract

The invention discloses an RNA library building method and a kit, and relates to the field of gene sequencing. The method comprises the following steps of: 1) synthesizing First cDNA to form a DNA / RNAhybrid chain; 2) breaking the First cDNA hybrid chain by using streptavidin magnetic beads immobilized with a Tn5 transposase complex, and connecting a joint; and 3) supplementing gaps of the fragmented hybrid chains and enriching a complete library through PCR amplification. According to the method provided by the invention, the library building process is simple to operate, and the RNA librarybuilding process can be completed within two hours at the soonest; moreover, the input amount of the initial RNA is relatively flexible, the constraint that the traditional Tn5 transposase amount onlyaims at the input amount of specific RNA to establish a library is broken through, the dependence of the transposase on template input is reduced, and 10ng-1[mu]g of RNA with different amounts can beinput according to own requirements.

Description

technical field [0001] The invention relates to the technical field of gene sequencing, in particular to a method for building an RNA library. Background technique [0002] High-throughput sequencing technology is abbreviated as NGS, which can simultaneously determine millions of DNA molecular sequences; its sequencing process includes sample extraction, library construction, sequencing and result analysis, and library construction includes RNA library construction and DNA library construction. The traditional RNA library construction is through high temperature and Mg after mRNA enrichment 2+ Interruption, first cDNA synthesis, second cDNA synthesis, purification, double-stranded cDNA fragment end repair and filling, double-stranded cDNA 3' end adding A, double-stranded cDNA fragment adding adapter, library amplification and other steps. This method takes a long time to build a database, the steps are complicated, and it is easy to make mistakes. [0003] In addition, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6806
CPCC12Q1/6806C40B50/06C12Q2563/143C12Q2563/149C12Q2531/113
Inventor 冯延叶李艳玲郭仕妹赖煦卉孙大鹏
Owner 上海英基生物科技有限公司
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