Construction method of RNA library for enriching original transcript information and application thereof

A construction method and transcript technology, applied in the field of RNA library construction, can solve the problems of lack of original transcript information, sequencing information results, etc.

Active Publication Date: 2016-04-20
BGI SHENZHEN CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But using this method, the obtained mRNA does not contain many other poly(A)-transcripts that can be used for transcriptional analysis
Moreover, the loss of original transcript information often

Method used

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  • Construction method of RNA library for enriching original transcript information and application thereof
  • Construction method of RNA library for enriching original transcript information and application thereof
  • Construction method of RNA library for enriching original transcript information and application thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0086] The construction of embodiment 1 RNA library

[0087] Specific experimental steps (see figure 1 Process steps shown in ):

[0088] 1. RNaseH purification of total RNA

[0089] 1.1 Take 1ug or 3ug of total RNA and anneal to oligoDNA

[0090]

[0091] 1.2 React at 95°C for 2 minutes on a PCR machine; cool down gradually, from 0.1°C to 22°C every 1 second; react at 22°C for 5 minutes, and place on ice quickly.

[0092] 1.3 RNaseH enzyme digestion

[0093]

[0094] React at 37°C for 30min

[0095] 1.4 DNaseI enzyme digestion

[0096]

[0097] 1.5 After the reaction, purify with RNAcleanXP magnetic beads and dissolve in 10ul nuclease-free water.

[0098] 2. RNA Fragmentation

[0099] Add 3 μL of 5×first-strand buffer to the eluate in the previous step, at 94°C for 10 min, and place on ice immediately.

[0100] 3. Reverse Transcribed First-Strand Synthesis

[0101] 3.1 Add 0.5 μL N6primer (0.1 μg / μL) to the RNA obtained in the previous step.

[0102] 3.2 Re...

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Abstract

The invention provides a construction method of a high-quality RNA library for enriching original transcript information by using RNase H and selectively removing lots of rRNA and blood globulin and an application thereof. The RNA library constructed by the method has very low rRNA residual volume (which is only 1/500 of residual volume in a conventional oligo(dT) method), and has very high gene coverage.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a method for constructing an RNA library enriched with original transcript information and its application. Background technique [0002] With the application and development of high-throughput sequencing technology, RNA sequencing technology is becoming more and more mature. Usually, when we construct RNA libraries, especially RNA-seq libraries, transcriptome libraries, and strand-specific transcription profile libraries, we need to perform mRNA purification on TotalRNA. Since the most abundant RNA in human cells is rRNA, accounting for about 82% of the total RNA, and the proportion of mRNA is only 4%. At present, the oligod(T) method is used to capture mRNA, relying on oligo(dT) to separate poly(A )+RNA to achieve the purpose of screening out a large amount of rRNA to obtain purified mRNA. But using this method, the obtained mRNA does not contain many other...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/08
Inventor 纪晓钧郭晶祝珍珍耿春雨李计广章文蔚蒋慧
Owner BGI SHENZHEN CO LTD
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