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Kit for constructing human single-cell TCR sequencing library and application of kit

A technology for sequencing libraries and single cells, applied in the field of molecular biology, can solve the problems of expensive sequencing of single-cell immune libraries and high requirements for cell activity, and achieve the effect of solving the problem of throughput and wide application prospects.

Pending Publication Date: 2021-06-25
SUZHOU GENO TRUTH BIOTECHNOLOGY CO LTD
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AI Technical Summary

Problems solved by technology

However, the 10X Genomics platform has limitations in the number of isolated single cells and requires high cell viability
At the same time, 10X Genomics-based single-cell immune repertoire sequencing is expensive

Method used

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  • Kit for constructing human single-cell TCR sequencing library and application of kit
  • Kit for constructing human single-cell TCR sequencing library and application of kit
  • Kit for constructing human single-cell TCR sequencing library and application of kit

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Embodiment Construction

[0035] An aspect of the embodiments of the present invention provides a kit for constructing a human single-cell TCR sequencing library, which includes:

[0036] A microfluidic chip, at least for capturing a single human T cell and packaging it to generate a water-in-oil reaction droplet, the water-in-oil reaction droplet includes an oil phase and a cell liquid phase wrapped by the oil phase, and The water-in-oil reaction droplets contain RNA reverse transcription components and cell lysis reagents;

[0037] oils, cell lysis reagents for forming said oily phase;

[0038] Cell tags, including deformable beads and molecular tags attached to the deformable beads, the molecular tags used to identify cells;

[0039] RNA reverse transcription assembly, comprising reverse transcription primer shown in SEQ ID NO:5, RNA reverse transcriptase, RNA reverse transcriptase inhibitor and RNA reverse transcription buffer;

[0040] cDNA pre-amplification components, comprising cDNA pre-ampli...

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Abstract

The invention discloses a kit for constructing a human single-cell TCR sequencing library and application of the kit. A method for constructing the human single-cell TCR sequencing library based on the kit comprises the following steps: capturing and packaging single cells by using a micro-fluidic chip; and successively conducting RNA reverse transcription, cDNA pre-amplification, primary amplification, secondary amplification, fragmentation, amplification, purification and the like. When the kit and the human single-cell TCR sequencing library are applied to sequencing of an immune repertoire of immune cells, 500-30000 cells can be separated in a single experiment, so the flux problem of the unicellular immune repertoire is fundamentally solved; and the kit and the human single-cell TCR sequencing library have wide application prospects in the fields of tumor microenvironment, infectious diseases, rejection after organ transplantation, immunotherapy and the like.

Description

technical field [0001] The present invention relates to a kit, in particular to a method for constructing a human-derived single-cell TCR (T cell receptor) sequencing library, a kit for constructing a human-derived single-cell TCR sequencing library, and a human-derived TCR sequencing method, belonging to field of molecular biology. Background technique [0002] The immune repertoire (immune repertoire, IR) refers to the sum of all functionally diverse T cells and T cells in the circulation of an individual at any given time. Traditional immune repertoire research techniques can only detect the sequence information of TCR (T cell receptor) and one chain of TCR, while single-cell immune repertoire sequencing can simultaneously obtain TCR α chain and β chain, TCR heavy chain and light chain and Combined information within a cell. [0003] Single-cell immune repertoire sequencing technology is a new technology for high-throughput sequencing of adaptive immune receptor reperto...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/6869
CPCC12Q1/6869C40B50/06C12Q2565/629C12Q2563/159C12Q2521/107C12Q2535/122
Inventor 张惠丹赵洪玉
Owner SUZHOU GENO TRUTH BIOTECHNOLOGY CO LTD
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