Preparation method of high-throughput sequencing library for miRNA

A sequencing library and high-throughput technology, applied in the field of high-throughput sequencing library preparation, can solve problems such as difficulty in satisfying accurate quantitative analysis, lack of accurate quantitative analysis of sequencing libraries, and inability to directly use library construction and quantitative analysis.

Pending Publication Date: 2020-12-01
上海京房生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These commercial kits are all aimed at miRNA library construction, and the experimental procedures are generally similar, but the constructed sequencing libraries are not accurate and quantitative, which is difficult to meet the needs of accurate quantitative analysis
The unique molecular identifiers (UMI) method used in single-cell sequencing has been reported by Saiful Islam et al. (Nature Methods, 11(2), 2014: 163-166), but it cannot be directly used in routine samples. RNA library construction and quantitative analysis
At present, there is no report of a fully quantitative library preparation technology for RNA, especially miRNA, from routine samples, so it cannot meet the actual needs of precision medicine and quantitative biology
In summary, there is still a lack of a high-throughput sequencing library preparation method for the precise quantification of miRNAs in routine samples.

Method used

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  • Preparation method of high-throughput sequencing library for miRNA
  • Preparation method of high-throughput sequencing library for miRNA
  • Preparation method of high-throughput sequencing library for miRNA

Examples

Experimental program
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Effect test

Embodiment 1

[0027] 3' end plus connector

[0028] 1. Dilute a total of 10 ng of total RNA extracted from HeLa cells with ultrapure water (no DNase and RNase, the same below) to a total volume of 5 μl, and place it in a 200 μl thin-walled PCR tube;

[0029] 2. Add 1 μl adapter RA3, mix well, incubate at 70°C for 2 minutes, and immediately place on ice to cool;

[0030] 3. Add 2 μl HML (Ligation Buffer), 1 μl RNase Inhibitor, 1 μl T4RNA Ligase, mix well, and incubate at 28°C for 1 hour;

[0031] 4. Add 1 μl of STP (Stopoligo solution with a concentration of 10 mM) and mix well, incubate at 28°C for 15 minutes; add a linker at the 5' end

[0032] 5. Take a new PCR tube and add 1.1 μl adapter RA5 (where S2 is a degenerate base N with a length of 10 10 , S3 is selected from ACGA);

[0033] 6. Degenerate base N 10 The concentration was 10 μM, incubated at 70°C for 2 minutes, and placed on ice immediately after the reaction;

[0034] 6. Add 1.1 μl 10mM ATP, then add 1.1 μl T4RNA ligase and ...

Embodiment 2

[0055] 3' end plus connector

[0056] 1. Dilute the total RNA extracted from A549 cells with a total amount of 1 μg to a total volume of 5 μl with ultrapure water, and place it in a 200 μl thin-walled PCR tube;

[0057] 2. Add 1 μl adapter RA3, mix well, incubate at 70°C for 2 minutes, and immediately place on ice to cool;

[0058] 3. Add 2 μl HML (Ligation Buffer), 1 μl RNase Inhibitor, 1 μl T4RNA Ligase2, Deletion Mutant, mix well, and incubate at 28°C for 1 hour;

[0059] 4. Add 1 μl of STP (10mM Stop oligo solution) and mix well, incubate at 28°C for 15 minutes; add adapter at 5' end

[0060] 5. Take a new PCR tube and add 1.1 μl adapter RA5 (where S2 is a degenerate base N with a length of 15 15 , the concentration of N15 is 10 μM, S3 is selected from CCGA), incubated at 70°C for 2 minutes, and placed on ice immediately after the reaction;

[0061] 6. Add 1.1 μl 10mM ATP, then add 1.1 μl T4RNA ligase and mix well;

[0062] 7. Add 3 μl of the above Mix to the reaction ...

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Abstract

At present, a special high-throughput sequencing library preparation method for accurately quantifying miRNA in a conventional sample does not exist; therefore, the invention provides a preparation method of a high-throughput sequencing library for the miRNA, which is accurate and quantitative; the method mainly comprises the following steps: mixing a certain amount of RNA sample with an adaptor RA3 to carry out ligation reaction, then carrying out ligation reaction with an adaptor RA5, then mixing with a reverse transcription primer RT Primer specifically bound to the adaptor RA3, and carrying out reverse transcription reaction to obtain a DNA first strand; then mixing with a universal primer RNA primer 1 specifically bound to a corresponding region of the adaptor RA3 and a specific primer RNA primer index specifically bound to a corresponding region of the adaptor RA5, and carrying out PCR reaction to obtain an amplification product; and finally, carrying out 6% gel electrophoresis on an amplification product, cutting a required target DNA fragment and recovering to obtain the prepared sequencing library, and carrying out length range detection and concentration quantification toobtain the sequencing library which can be used for Ilunima high-throughput sequencing platform direct sequencing.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically, relates to a method for preparing a high-throughput sequencing library for miRNA (English full name is microRNA, Chinese name is microRNA). Background technique [0002] miRNA is a kind of endogenous small RNA with a length of about 22 nucleotides, which has a variety of important regulatory functions in cells. At present, research on the sequence composition and function of miRNA has gradually become a hotspot. The mainstream miRNA high-throughput library preparation kits on the market, such as Illumina’s TruSeqSmall RNA library preparation kit, and NEB’s Small RNA library preparation kit developed for Illumina sequencing platforms Box (NEB Multiplex Small RNA LibraryPrep Set for Illumina), etc. These commercial kits are all designed for miRNA library construction, and the experimental procedures are generally similar. However, the constructed sequencing libraries are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C40B40/06C12Q1/6869
CPCC40B50/06C40B40/06C12Q1/6869C12Q2531/113C12Q2535/122
Inventor 李华郭子文谢跃华
Owner 上海京房生物科技有限公司
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