High-throughput single-cell transcriptome sequencing method and kit

A transcriptome sequencing and single-cell technology, applied in the field of single-cell sequencing, can solve the problems of inability to carry out large-scale research work, difficult detailed analysis of different cell subpopulations, constrained throughput, cost, etc., to reduce mutual contamination of microbeads , Convenient single-cell sequencing, and low-cost effects

Pending Publication Date: 2020-01-14
MGI TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In general, the existing single-cell separation or single-cell transcriptome sequencing technologies are limited by issues such as throughput and cost, and many large-scale research work cannot be carried out
On the one hand, low throughput m

Method used

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  • High-throughput single-cell transcriptome sequencing method and kit
  • High-throughput single-cell transcriptome sequencing method and kit
  • High-throughput single-cell transcriptome sequencing method and kit

Examples

Experimental program
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Embodiment

[0058] 1. Prepare reagents

[0059] (1) Cell resuspension

[0060] Prepare 5 mL of PBS-BSA with a final concentration of 0.01% BSA; wash the H1975 cell line with PBS-BSA, and resuspend the cells in PBS-BSA for later use.

[0061] (2) Cleavage and reverse transcription reaction reagent preparation

[0062] 1mL of lysis and reverse transcription reaction reagents include: 10% Triton-X 10μL, 0.1M DTT 125μL, RnaseOUT 75μL, 10Mm each dNTPs 50μL, 5×RT buffer400μL, Super Script II ReverseTranscriptase 75μL, Template Switch Oligo 20μL, Rnase-free H 2 O 265 μL.

[0063] Wherein, Template Switch Oligo is the sequence shown in SEQ ID NO.5,

[0064] SEQ ID NO.5: 5'-AAGCAGTGGTATCAACGCAGAGTGAATGGG-3'

[0065] In Template Switch Oligo, the penultimate 2nd and 3rd bases are modified with riboguanosine, and the last base is modified with LNA.

[0066] (3) Microbead resuspension

[0067] In this example, the microbeads have primer sequences with poly(T) random sequences, which can capture...

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Abstract

The invention discloses a high-throughput single-cell transcriptome sequencing method and a kit. The high-throughput single-cell transcriptome sequencing method includes the steps that a droplet generation system is adopted to encapsulate a single cell and labeled microbeads in a droplet, and reverse transcription is performed in the droplet. According to the high-throughput single-cell transcriptome sequencing method, a throughput of 9,000 cells which is equivalent to 10 x genomics can be achieved, after the droplet is demulsified, the microbeads are less contaminated with each other, and theproportion of valid data is increased. According to the high-throughput single-cell transcriptome sequencing method, reverse transcription is performed in the droplet, fewer reagents are required, and the cost is low. In the preferred scheme of the high-throughput single-cell transcriptome sequencing method, a SMART template conversion technology is adopted in reverse transcription, and productscan be directly used for subsequent Tn5 library construction and BGISeq-500 platform sequencing without library conversion; and multiple amplification and introduction of deviations are avoided, a droplet-based microfluidics platform is used in conjunction with a BGISeq-500 sequencing platform, and large-scale single-cell sequencing is simplified and facilitated.

Description

technical field [0001] This application relates to the field of single-cell sequencing, in particular to a high-throughput single-cell transcriptome sequencing method and kit. Background technique [0002] The mechanism of disease occurrence and their detection and prevention are mainly analyzed through the detection of cell genome variation and abnormal expression of transcriptome. Single-cell sequencing technology can reveal the complex heterogeneity of cells in tissues and provide accurate information for the diagnosis and treatment of diseases. Constrained by the processing throughput and cost of single-cell sequencing technology, many large-scale research work cannot be carried out. The combination of microfluidic technology and single-cell sequencing technology can solve these problems well; at the same time, single-molecule sequencing technology can avoid Errors caused by nucleic acid amplification have great application prospects and demands in basic scientific rese...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2563/159C12Q2535/122C12Q2563/185
Inventor 赵星金皓玄李罗权赵小莹冯太青齐晓娟周清李贵波李计广王磊李阳
Owner MGI TECH CO LTD
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