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Kit and library construction method for constructing single-cell transcriptome sequencing library

A technology for transcriptome sequencing and construction methods, which is applied in the field of kits for constructing single-cell transcriptome sequencing libraries, can solve the problems of low effective data ratio and adverse effects of data analysis, and achieve the effect of increasing the effective data ratio

Active Publication Date: 2019-05-21
ANNOROAD GENE TECH BEIJING +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when using, for example, an Illumina high-throughput sequencer for sequencing, the proportion of valid data obtained from the high-throughput sequencing library constructed by this method is low, which will adversely affect subsequent data analysis

Method used

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  • Kit and library construction method for constructing single-cell transcriptome sequencing library
  • Kit and library construction method for constructing single-cell transcriptome sequencing library
  • Kit and library construction method for constructing single-cell transcriptome sequencing library

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Experimental program
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Embodiment

[0070] The present invention will be described in further detail below in conjunction with the examples. It should be understood that the specific embodiments described here are used to explain the present invention, not to limit the present invention.

[0071] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0072] The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1

[0073] Example 1 Using the library construction method of the present invention to construct a single-cell transcriptome sequencing library

[0074] 1. Reverse transcription and amplification of cDNA

[0075] 1.1 Single cells were collected into 0.2 mL thin-walled PCR tubes with a volume of less than 0.5 μL, which contained 2 μL of mildly hypotonic lysis buffer. The composition of the lysate is as follows:

[0076] Triton X-100

0.2％

Sigma

RNase inhibitor

2U / μL

Enzymatics

[0077] 1.2 Cell lysate was mixed with 1 μL oligo-dT primer and 1 μL dNTP mix, denatured at 72°C for 3 minutes and placed on ice immediately. Then add 7 μL of a chain reaction system, the reaction system contains the following:

[0078]

[0079]

[0080] oligo-dT primers:

[0081] (10 μM, 5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′)

[0082] TSO:

[0083] (10 μM, 5′-AAGCAGTGGTATCAACGCAGAGTACrGrG+G-3′)

[0084] After the addition, the reverse transcription reaction pro...

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Abstract

The invention provides a method for constructing a single cell transcriptome sequencing library. A DNA (Deoxyribonucleic acid) fragment library for sequencing, which is established in an earlier stage, is subjected to enzyme digestion by using a RsaI restriction enzyme, and an enzyme digestion product is subjected to PCR (Polymerase Chain Reaction) amplification, so that a proportion of effective data for sequencing the single cell transcriptome sequencing library can be remarkably increased.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for constructing a single-cell transcriptome sequencing library, a single-cell transcriptome sequencing method and a kit for constructing a single-cell transcriptome sequencing library. Background technique [0002] Genome-wide transcriptome analysis is widely used, and the early method is to obtain RNA from a large number of tissue samples for sequencing. However, this traditional method relies on the overall analysis of the gene expression of millions of cells at a time, which often masks biologically meaningful gene expression differences of some specialized cells in specific tissues. Also, in healthy tissues or diseased tissues such as tumors, certain cells are so rare that they cannot be analyzed by any other technique except single-cell methods. [0003] Single-cell gene expression analysis overcomes these limitations and can be used to mine gene regul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/6806C12Q1/6869C40B50/06
Inventor 柳青王占东邵长超曹善柏刘涛杨洁玄兆伶李大为梁峻彬陈重建
Owner ANNOROAD GENE TECH BEIJING