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Method and kit capable of removing ribosomal RNA with high efficiency for construction of transcriptome sequencing library

A transcriptome sequencing and library technology, applied in the biological field, can solve the problems that affect the detection and analysis of mRNA or LncRNA, fail to effectively remove rRNA kits, rRNA residues, etc., so as to improve the efficiency of sequencing detection and reduce the amount of rRNA residues. , the effect of simple operation

Active Publication Date: 2018-04-10
GUANGZHOU RIBOBIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for samples with an initial total RNA amount of 1ng-100ng, there is no kit that can effectively remove rRNA and construct a transcriptome library.
This limitation may lead to serious rRNA residues and data redundancy in the construction of transcriptome sequencing libraries from samples such as plasma, serum, and Exosome, which will affect the subsequent detection and analysis of mRNA or LncRNA

Method used

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  • Method and kit capable of removing ribosomal RNA with high efficiency for construction of transcriptome sequencing library
  • Method and kit capable of removing ribosomal RNA with high efficiency for construction of transcriptome sequencing library
  • Method and kit capable of removing ribosomal RNA with high efficiency for construction of transcriptome sequencing library

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: gRNA design density

[0037] 1. Building a database

[0038] 1. Cell culture

[0039] Use Gibco's DMEM and 10% FBS serum at 37 °C and 5% CO 2 The 293T cells are cultured under culture conditions, and after 2 days of normal culture, the cell density is about 60-80% (40,000-50,000).

[0040] 2. RNA extraction

[0041] Total RNA was extracted according to Magen's RaPure Total RNA Kit instructions.

[0042] 3. RNA library construction:

[0043] NEBNext RNA Ultra-Rapid Library Preparation Kit-Illumina was used for library construction with 10ng starting amount (cell RNA), the main steps were: RNA fragmentation (the average fragment length after fragmentation was 200bp); first-strand cDNA synthesis; Second-strand cDNA synthesis and purification; end repair and purification; 3' end addition and purification; adapter ligation and purification; PCR amplification and purification, the cDNA gene library was obtained.

[0044] 2. Library for rRNA depletion

[0045...

Embodiment 2

[0077] Embodiment 2: Cas9 dosage

[0078] 1. RNA library construction

[0079] Concrete method is the same as embodiment one.

[0080] 2. RNA library for rRNA removal

[0081] Concrete method is the same as embodiment one.

[0082] Among them, the design density of gRNApool is 200bp / strip; the working concentration of Cas9 is 30nM, 300nM, 3μM, 30μM and 300μM for experiments.

[0083] 3. On-machine detection of RNA library

[0084] Concrete method is the same as embodiment one.

[0085] 4. Results Analysis

[0086] The results of sequencing analysis showed that the rRNA residues in the rRNA-removed library were all below 30%; finally, the rRNA residues at the dosage of 300nM Cas9 were the least, accounting for only 3.46%, and the number of genes and transcript arrays that could be analyzed were large, respectively. For 26470 and 66887.

[0087]

Embodiment 3

[0088] Embodiment three: reaction time

[0089] 1. RNA library construction

[0090] Concrete method is the same as embodiment one.

[0091] 2. RNA library for rRNA removal

[0092] Concrete method is the same as embodiment one.

[0093] The design density of the gRNA pool is 200bp / strip, the working concentration of Cas9 is 300nM, and the rRNA removal treatment is performed at different times of 5min, 30min, 1h, 2h and 5h respectively.

[0094] 3. On-machine detection of RNA library

[0095] Concrete method is the same as embodiment one.

[0096] 4. Results Analysis

[0097] The results of sequencing analysis showed that the rRNA residues in the library after rRNA removal were all below 20%; when the rRNA removal reaction time was 1 h, the rRNA residues were the least, accounting for only 3.46%, and the number of genes and transcript arrays that could be analyzed were large. 26470 and 66887 respectively.

[0098]

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Abstract

The invention discloses a method for construction of a transcriptome sequencing library. The method comprises the following steps: providing a DNA molecule containing to-be-removed rRNA; carrying outdesigning on account of a DNA sequence of the to-be-removed rRNA so as to obtain a Crispr RNA pool; and allowing a DNA sample to contact with the Crispr RNA pool and Crispr protein so as to obtain a sample compiled by a Crispr system and obtain the transcriptome sequencing library and a kit used for construction of the transcriptome sequencing library. The method and the kit provided by the invention can remove a large number of redundant RNA like rRNA with high efficiency, enrich other molecules in the sample, improve the data quality and the effective data ratio of initial transcriptome sequencing, and reduce the cost of sequencing.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method and a kit for efficiently removing ribosomal RNA for constructing a transcriptome sequencing library. Background technique [0002] Ribosomal RNA (rRNA) constitutes the vast majority of cellular RNA content, accounting for approximately 80%-95% of intracellular RNA content. The high abundance of rRNA complicates the analysis of other related target molecules in the sample, such as transcriptome sequencing (RNA-seq) analysis, microarray gene expression analysis, etc.; especially in studies with less target molecule content, rRNA becomes Huge background pollution. Therefore, usually when constructing an RNA library (for example, when constructing a transcriptome library), it is necessary to purify the total RNA from mRNA (the ratio is about 10%). [0003] Transcriptome sequencing can comprehensively and quickly obtain almost all transcript sequence information of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06
CPCC12N15/1093C40B50/06
Inventor 张必良克雷格·梅洛
Owner GUANGZHOU RIBOBIO
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