Method for rapidly constructing strand-specific RNA high-throughput sequencing library

A sequencing library, specific technology, applied in the field of genetic engineering, can solve the problems of high material and labor cost, short time, complicated operation, etc., and achieve the effect of high efficiency, short time, and simplified operation process

Inactive Publication Date: 2018-07-06
HUNAN YEARTH BIOTECHNOLOGICAL CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the problems existing in the prior art, the present invention provides a method for rapidly constructing a strand-specific RNA hi

Method used

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  • Method for rapidly constructing strand-specific RNA high-throughput sequencing library
  • Method for rapidly constructing strand-specific RNA high-throughput sequencing library
  • Method for rapidly constructing strand-specific RNA high-throughput sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] refer to Figure 2-3 In this embodiment, the method for rapidly constructing an exosome RNA library is carried out according to the following steps:

[0030] (1) Using a fragmentation reagent to fragment the RNA sample;

[0031] Take 4ml of plasma to separate exosomes and extract RNA according to the instructions of exoRNeasy Serum / Plasma Maxi Kit (77064, QIAGEN);

[0032] Prepare the reaction system in Table 1 in a PCR tube, process it in a thermal cycler at 85°C for 6 minutes, and put it on ice immediately;

[0033] Table 1 fragmentation reaction system

[0034] components

Volume (μL)

RNA

13

RT-Buffer

4

1uM RT-HEX

1

total capacity

18

[0035] The RT-HEX nucleic acid sequence is: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNN;

[0036] (2) After step (1), directly add reverse transcription primers, template jump primers and reverse transcription reagents to realize reverse transcription from RNA to DNA and add seq...

Embodiment 2

[0069] This embodiment is to compare the rapid chain-specific RNA library construction method with the traditional RNA library construction method. The specific steps are:

[0070] 1. Rapid strand-specific RNA library construction method

[0071] Remove the rRNA sample and perform rapid construction of a strand-specific RNA library according to the method in Example 1.

[0072] 2. Traditional RNA library construction method

[0073] Take the same rRNA-depleted sample and perform library construction according to the instructions of VAHTS Total RNA-seq (H / M / R) Library Prep Kit for Illumina (NR603, Novizyme, Nanjing), including the following steps:

[0074] 2.1 Fragmentation and purification of RNA by temperature control and metal ions;

[0075] 2.2 First-strand cDNA synthesis;

[0076] 2.3 Second strand cDNA synthesis;

[0077] 2.4 Use VAHTS DNA Clean Beads to purify the product of step 2.2.3;

[0078] 2.5 End repair, purification;

[0079] 2.6 Add dA-Tailing;

[0080] 2...

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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for rapidly constructing a strand-specific RNA high-throughput sequencing library. The methodprovided by the invention completes construction of an RNA library in only four steps as follows: fragmentation, RT and PCR enrichment and library purification. Compared with a traditional RNA libraryconstruction method, the method provided by the invention simplifies operation flows, shortens time, has high efficiency and can produce a high-quality strand-specific RNA-seq library by only two enzymes.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a method for rapidly constructing a strand-specific RNA high-throughput sequencing library. technical background [0002] In recent years, with the successive introduction of second-generation high-throughput sequencers such as Illumina, Roche 454, and SOLiD, the cost of sequencing has dropped significantly. Gain deeper insight into the interactions between the genome, transcriptome, and proteome. RNA sequencing (RNA-seq) has become an indispensable tool in biology and medicine. RNA sequence information, as a representation of the body's genes at the transcriptional level, provides important support for the application of methods such as disease diagnosis and gene therapy. [0003] At present, RNA sequencing needs to construct a library. Taking the RNA sample with rRNA removed as an example, the traditional RNA library construction process is as shown in the attache...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6869C40B50/06
CPCC12Q1/6806C12Q1/6869C40B50/06
Inventor 唐琼沈欢徐根明潘艺赵谦
Owner HUNAN YEARTH BIOTECHNOLOGICAL CO LTD
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