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Method and kit for labeling nucleic acid molecules

A technology of nucleic acid fragments and double-stranded nucleic acids, which is applied in the field of high-throughput single-cell transcriptome sequencing and transcriptome sequencing, can solve the problems of high cost of library construction, high empty load rate of micro-reaction system, and low sample throughput, achieving The effect of reducing adverse effects and facilitating commercial application

Pending Publication Date: 2022-02-08
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This means that the number of droplets containing reagents and magnetic beads produced by a single reaction is about 100,000, and the effective utilization rate is less than 10%.
[0014] At present, the main disadvantages of the commercial methods of 5'-end library construction technology for transcriptome sequencing (such as the 5'-end library construction solution developed by 10X Genomics) are: low cell throughput, high emptying rate of the microreaction system, and sample Low throughput, high cost of library construction
The more cells on the machine, the more "false single cell" data needs to be removed, and the higher the wasted sequencing cost
Therefore, this 5'-end library construction scheme still cannot solve the problems of low cell throughput and high empty load rate of the micro-reaction system, and the cost of transcriptome sequencing is still high
[0015] In summary, the existing high-throughput single-cell transcription library construction technology (especially the 5' end library construction technology) still has the following defects: low cell throughput, high empty rate of the micro-reaction system, and high cost of library construction

Method used

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  • Method and kit for labeling nucleic acid molecules
  • Method and kit for labeling nucleic acid molecules
  • Method and kit for labeling nucleic acid molecules

Examples

Experimental program
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Effect test

Embodiment 1

[0270] Example 1: Preparation of TN5 transposase complexes labeled with single-end specific oligonucleotides

[0271] The reagent / instrument information used in this embodiment is as shown in Table 2:

[0272] Table 2 Reagents and Instruments

[0273]

[0274] Note: "5Phos" means that the 5' end contains phosphorylation modification; "3ddC" means that the 3' end is cytosine dideoxyribonucleotide.

[0275] experimental method:

[0276] 1. Preparation of transposons

[0277] (1) Transposon adapter 1 (SEQ ID NO:1) and tagged transposon adapter 2 (SEQ ID NO:2) were used respectively The Annealing buffer in the Tagment Enzyme kit was dissolved to 100uM, and then the transposon adapter 1 and 96 kinds of tagged transposon adapters 2 (the tag sequences are shown in SEQ ID Nos: 3-98) were mixed at a ratio of 1:1 Mix by volume. The mixing steps are as follows: take 10 ul of joint 1 and joint 2 respectively, mix thoroughly in a 96-well PCR plate, cover the 96-well PCR plate with...

Embodiment 2

[0287] Example 2: Preparation of single cell nuclei

[0288] The reagent / instrument information used in this embodiment is as shown in Table 3:

[0289] Table 3 Reagents and Instruments

[0290] Reagent / instrument name brand Item No. 1x PBS Gibco 14190-094 1M Tris-HCl pH7.5 Jena Bioscience BU-125S 1M MgCl 2

Merck Millipore 20-303 5M NaCl Sangong B548121-0100 Tween-20 Sigma P7949-500ML IGEPAL CA-630 Sigma I8896-50ML BSA bovine serum albumin Sigma A8806-5 Digitonin Abcam ab141501 SUPERase-In RNase Inhibitor Thermo Fisher Scientific AM2696 nuclease-free water Invitrogen AM9932 Flowmi Cell Strainer, 40μM Bel-Art H13680-0040 1.5ml DNA low adsorption tube Eppendorf 30108051 HEK293T cell line Chinese Academy of Sciences Cell Bank GNHu17 Hela cell line Chinese Academy of Sciences Cell Bank TCHu187 K562 cell line Chinese Academy of Sciences Cel...

Embodiment 3

[0296] Example 3: Permeabilization of Single Cell Suspension

[0297] The reagent / instrument information used in this embodiment is as shown in Table 4:

[0298] Table 4 Reagents and Instruments

[0299] Reagent / instrument name brand Item No. Methanol Fisher Chemical M / 4000 / 17 1x PBS Gibco 14190-094 BSA bovine serum albumin Sigma A8806-5 SUPERase-In RNase Inhibitor Thermo Fisher Scientific AM2696 nuclease-free water Invitrogen AM9932 Flowmi Cell Strainer, 40μm Bel-Art H13680-0040 1.5ml DNA low adsorption tube Eppendorf 30108051 centrifuge Thermo Fisher Scientific Micro21R Automatic fluorescence cell counter LUNA LUNA FL

[0300] experimental method:

[0301] 1. Prepare fresh sample dilution (1x PBS, add 1% BSA, 1% SUPERase-In RNase Inhibitor);

[0302] 2. According to the needs of the experiment, fresh tissue, fresh cell lines, fresh blood samples, primary cells, and frozen cell sam...

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Abstract

The invention discloses a method and a kit for labeling nucleic acid molecules. The invention relates to a method for processing a cell or a cell nucleus to generate a group of nucleic acid fragments, and a method for generating labeled nucleic acid molecules by using the group of nucleic acid fragments, constructing a nucleic acid molecule library for transcriptome sequencing, or performing high-throughput sequencing on a single cell transcriptome. In addition, the invention also relates to a nucleic acid molecule library constructed by using the method, and a kit for implementing the method.

Description

technical field [0001] The present application relates to transcriptome sequencing, especially the technical field of high-throughput single-cell transcriptome sequencing. In particular, the present application relates to a method of treating cells or nuclei to generate a population of nucleic acid fragments, and utilizing said population of nucleic acid fragments to generate labeled nucleic acid molecules, construct a library of nucleic acid molecules for transcriptome sequencing, or, for Methods for high-throughput sequencing of single-cell transcriptomes. In addition, the present application also relates to a library of nucleic acid molecules constructed by the method, and a kit for implementing the method. Background technique [0002] Cells are the basic structural and functional units of organisms, and the study of their function and heterogeneity has always been a major challenge in the field of biology. The development of single-cell omics sequencing technology has...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6869C40B50/06C40B40/06
CPCC12Q1/6806C12Q1/6869C40B50/06C40B40/06C12Q2535/122C12N15/1096C12N15/1065C12Q2521/107C12Q2563/179C12Q2563/149C12Q2531/113
Inventor 蒋岚李芸
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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