Sequencing method for single cell transcriptome

A single-cell, transcriptome technology, applied in the biological field, can solve problems such as undetectable, no total RNA or long non-coding RNA single-cell RNA sequencing methods

Active Publication Date: 2019-07-05
复旦大学泰州健康科学研究院
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  • Abstract
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Problems solved by technology

[0008] However, all current single-cell RNA-Seq methods use an oligo(dT) method for reverse transcription of polyA-tailed RNA. This method can effectively get rid of ribosomal RNA (rRNA) interference, but cannot detect those without polyA Tailed RNA (except for circular circRNA sequencing mentioned above), such as long non-coding RNA without A tail, etc., there is no single-cell RNA sequencing method for total RNA or long non-coding RNA

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  • Sequencing method for single cell transcriptome
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  • Sequencing method for single cell transcriptome

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Embodiment 1

[0036] For the single-cell total RNA sequencing of five common cell lines, the specific implementation steps are as follows.

[0037] 1. Use 1% Trypsin to digest LNCaP, T24, PC3, HEK293t, and C2C12 cells for 10-15 minutes to a single cell state, resuspend in PBS, and absorb 6 cells of each type with a capillary tube, and then lyse them in CLB.

[0038] 2. PolyA polymerase reaction: configure the cell lysis system according to Table 1, first CLB and H 2 O, DTT, MnCl 2 , ATP mixed and reacted at 37°C for 3min, then added Poly(A) Polymerase and 0.5% BSA and reacted at 37°C for 5min or 15min. Prepare the PolyA Polymerase reaction solution according to Table 2, and place it on ice after reacting at 72° C. for 3 minutes.

[0039] Table 1 Cell Lysis System

[0040]

[0041] Table 2 PolyA Polymerase reaction liquid formula

[0042] SINGLE-CELL CUSTOM SMARTER 1T PAP reaction buffer 5μl dNTPs mix (10mM) 1.00μl oligo dT (10μM) 1.00μl ERCC spike-i...

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Abstract

The present invention belongs to the field of biotechnology and discloses a sequencing method for a single cell transcriptome. PAP/PGP is used to add poly A/G to a tail part of a RNA strand to increase RNA stability and at the same time produces an extended strand used for reverse transcription and cDNA synthesis. When effective RNA enrichment is carried out downstream, a small-fragment PCR non-specific product is purified by magnetic beads labeled with ribosomal DNA probes, a half-annealed splint hybridization is used to remove rRNA in a cDNA form having a size of 5s, 5.8s, 18s and 28s, and besides, a large amount of invalid Reads during sequencing are reduced. The sequencing method can conduct one-stop non-coding RNA, microRNA and mRNA total transcriptome sequencing of a single eukaryocyte, produces an expression matrix of the transcriptome and frees an application limitation that one cell can only perform one sequencing.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a single-cell transcriptome sequencing method, in particular to a single-cell total RNA or single-cell long-chain non-coding RNA sequencing method. Background technique [0002] Sequencing is a method of determining the sequence of a gene. The genes of each species control the expression of biological traits, and sequencing refers to the judgment of the sequence of genes. The first-generation DNA sequencing technology (also known as Sanger sequencing) was pioneered by Sanger et al. in 1975, and the first genome sequence (bacteriophage X174) was completed in 1977, with a full length of 5375 bases. After 30 years of practice and continuous improvement of technology and sequencing strategies (such as mapping and shotgun methods using different strategies), the first human genome map completed in 2001 was sequenced using the improved Sanger method Base. Although Sanger seq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2525/173C12Q2535/122C12Q2531/113
Inventor 陈兴栋朱嗣博庆涛金力
Owner 复旦大学泰州健康科学研究院
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