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Ultrahigh-flux single-cell chromatin transposase accessibility sequencing method

A transposase and chromatin technology, applied in the field of single-cell sequencing, can solve the problems of high cost, low contamination rate, low nuclear flux, etc., and achieve the effect of improving flux

Active Publication Date: 2021-11-05
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The single-cell ATAC platform based on microfluidics and micropores has high sensitivity and low contamination rate, but the analysis throughput is low. A single experiment can only process about 10,000 single-cell samples, and the cost is high
sci-ATAC-seq is limited by the number of split-pool combinations, the nuclear flux of the analysis is low and the contamination rate between cells is high
On the basis of the former, sci-ATAC-seq3 uses an untagged transposase to insert the transposon fragment and then uses two consecutive rounds of split-pool connection to add cell tags, which increases the number of combinations. The efficiency of the wheel connection sacrifices part of the sensitivity
At the same time, the nucleus pre-labeling system requires multiple rounds of split-pool process, resulting in low cell recovery efficiency (about 10%)

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0060] Embodiment 1: microplate preparation

[0061] According to the experimental scale (500,000 human 293T cells and 500,000 mouse 3T3 cells each), design the size of the microwell plate (the size of the well plate is 1.8cm×1.8cm), and etch the microwell on the silicon wafer as the initial mold, and the microwell is a cylinder Body shape, in which the depth of micropores is 60 μm, the diameter of micropores is 50 μm, and the distance between pores is 70 μm. Next pour polydimethylsiloxane (PDMS) on the silicon chip, take off PDMS after molding and become the second mold that has microcolumn on the plate, the microporous plate that final experiment uses is that concentration is 5% (mass ratio ) agarose (prepared with enzyme-free water), poured on the PDMS microcolumn plate after hot melting to condense and form, and the agarose plate at this time is a microwell plate with a certain thickness after being peeled off. When saving, add DPBS-EDTA mixture which is harmless to cells...

Embodiment 2

[0062] Example 2: Preparation of molecularly labeled magnetic beads

[0063] The magnetic beads were purchased from Suzhou Zhiyi Microsphere Technology Co., Ltd. (Product No. MagCOOH-20190725), the surface was coated with carboxyl groups, and the diameter was 45 μm. The preparation process of molecularly labeled magnetic beads is as follows: figure 1 As shown, there are 5 steps in total:

[0064] (1) Design the molecular marker sequence, divide the molecular marker sequence into three sections, and set a linker sequence between the adjacent two sections for connecting the adjacent two sections through PCR, and the first section starting from 5' includes the universal primer sequence and part of the cell tag sequence, the last segment contains part of the cell tag sequence, the entire molecular tag sequence, and the bridging complementary sequence. Except for the first segment, the rest of the sequences are complementary sequences of the corresponding sequence.

[0065] (2) T...

Embodiment 3

[0073] Example 3: Preparation of Specific Molecular Tag Transposase Embedding Complex

[0074] Tn5 transposase naked enzyme was purchased from Nanjing Novizan Biotechnology Co., Ltd. The transposase and embedding buffer were provided by (Vazyme) Tn5 Transposome (S111) kit produced by Nanjing Novizan Biotechnology Co., Ltd.

[0075] (1) The embedded nucleic acid sequence is shown in Table 2 below.

[0076] Table 2

[0077]

[0078]Among them, the specific molecular tag 10×N contained in the specific tag fragment is the core sequence of the cell tag sequence, and the core sequence corresponding to each Tn5 complex is different. choices, so a 10×N sequence can have 4 10 options. N represents any one of A / T / C / G, which is randomly synthesized.

[0079] (2) Annealing of 96 oligonucleotide sequences with specific molecular tags (P7 adapter fragment + Mosaic Ends fragment annealing to obtain a universal sequence, namely figure 2 The P7 Adapter-Mosaic Ends / Mosaic Ends in the ...

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Abstract

The invention discloses an ultrahigh-flux single-cell chromatin transposase accessibility sequencing method. The ultrahigh-flux single-cell chromatin transposase accessibility sequencing method comprises the following steps that firstly, transposition of a chromatin transposase accessibility genome sequence in a cell nucleus is carried out by using a specific molecular tag transposase embedding compound; one molecular marker microbead and one or more cell nucleuses are located in a separated space through a microwell plate technology or a microfluidic technology, the cell nucleuses are split under the action of a splitting solution, and a specific molecular tag sequence is connected with a molecular marker sequence on the molecular marker microbead with the help of a bridging primer; a large number of sequences are obtained through PCR amplification, and a sequencing library is constructed and obtained; and then high-throughput sequencing is carried out, specific genome openness information of millions of single cells can be obtained through one-time sequencing, the single cell sequencing throughput is greatly improved, and the development of a single-cell high-flux chromatin transposase accessibility sequencing technology based on a microwell plate is actively promoted.

Description

technical field [0001] The invention relates to the technical field of single-cell sequencing, in particular to an ultra-high-throughput single-cell chromatin transposase accessibility sequencing method. Background technique [0002] In the genome, most of the chromatin is tightly wound in the nucleus and is not transcriptionally active. The chromatin state is dynamically regulated in a cell-type-specific manner, and partially dense chromatin becomes loose in a specific cell state. These loose chromatin are called open chromatin or accessible chromatin. . By detecting the opening of cell chromatin, information about cell transcription regulation can be obtained, such as where transcription factors can bind to gene promoters, which genes in cells may be efficiently transcribed, etc. Commonly used detection methods are ATAC-seq, DNase-seq, MNase-seq, FAIRE-seq and ChIP-seq, etc. These methods are based on different principles to interrupt and mark open chromatin regions. Qu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869B29C67/20B29C39/02
CPCC12Q1/6869B29C67/20B29C39/021C12Q2531/113C12Q2535/122C12Q2563/143C12Q2563/149C12Q2563/179
Inventor 郭国骥陈海德张国栋杨蕾韩晓平
Owner ZHEJIANG UNIV
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