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A precise and quantitative atac-seq library preparation method and kit

A kit and library technology, applied in the field of accurate and quantitative ATAC-seq library preparation methods and kits, can solve problems such as increased data repetition rate, systematic errors, etc., and achieve the effects of simple operation and easy access to conditions

Active Publication Date: 2022-04-22
苏州京脉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These kits can satisfy different sources of plant samples, and satisfy scientific research applications to the greatest extent. However, there is currently no method for accurate and quantitative library preparation on the market. The experimental process of the library construction method in the conventional ATAC-seq library There are still systematic errors, especially for a small number of samples, it is necessary to increase the amount of sequencing to improve the accuracy of detection, and in this case the repetition rate of data will increase significantly, which limits the application of this method in precision medicine and disease prediction other needs

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  • A precise and quantitative atac-seq library preparation method and kit
  • A precise and quantitative atac-seq library preparation method and kit
  • A precise and quantitative atac-seq library preparation method and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Example 1: Plant samples

[0053] Preparation of the transposome complex:

[0054] (1) Thaw commercially purchased Tn5 Transposase on ice and shake well before use;

[0055] (2) The first adapter and the second adapter were annealed and hybridized with the single-stranded M to form a double-stranded adapter. The sequence of the single-stranded M was 5'-CTGTCTCTTATACACATCT-3, and the lengths of S2 and S6 were both 15 bp.

[0056] (3) Mix the Tn5 enzyme and the synthesized double-stranded adapter according to the molar ratio of 1:1, and incubate at room temperature for 1 hour to obtain the transposition complex.

[0057] Sequencing library preparation:

[0058] (1) Obtain Arabidopsis thaliana, wash it with ultrapure water for 3-5 times, wipe the surface of the leaf with a dust-free paper, use a pre-cooled mortar to crush the leaf tissue in liquid nitrogen, and freeze the leaf tissue Store in -80°C freezer for later use.

[0059] (2) Resuspend the tissue fragments ...

Embodiment 2

[0085] Example 2: Human samples

[0086] Preparation of the transposome complex:

[0087] (1) Thaw commercially purchased Tn5 Transposase on ice and shake well before use;

[0088] (2) The first adapter and the second adapter were annealed and hybridized with the single-stranded M to form a double-stranded adapter. The sequence of the single-stranded M was 5'-CTGTCTCTTATACACATCT-3, and the lengths of S2 and S6 were both 15 bp.

[0089] (3) Mix the Tn5 enzyme and the synthesized double-stranded adapter according to the molar ratio of 1:1, and incubate at room temperature for 1 hour to obtain the transposition complex.

[0090] Sequencing library preparation:

[0091] (1) Obtain 10,000 intact human hepg2 cells, centrifuge at 500×g at 4°C for 5 minutes, and discard the supernatant.

[0092] (2) Add 50μl pre-cooled lysis buffer (10mM Tris-HCl, pH 7.4, 10mM NaCl, 3mMMgCl 2 , 0.1% IGEPPAL CA-630). Incubate on ice for 5-10min. Immediately centrifuge at 500×g 4°C for 5 minut...

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Abstract

The present application provides an accurate and quantitative ATAC-seq library preparation method and kit, including two adaptors, the first adaptor is 5'-S1-S2-S3-S4-3', and the second adaptor is 5' -S5-S6-S7-S8-3', wherein, described S1 is the first sequencing primer, S2 is a random tag sequence, and the base of S2 is one or more of A, T, G, C random Combination, the number of bases of S2 is any integer of 1-95, S3 is a fixed sequence, the base sequences of described S4 and S8 are identical, are Tn5 transposon fixed sequence, S5 is the second sequencing primer, and S6 is Random tag sequence, the base of S6 is a random combination of one or more of A, T, G, and C, the number of bases of S6 is any integer of 1-100, and S7 is another fixed sequence, the The two adaptors are incubated with Tn5 enzyme to obtain a transposition complex; then the transposition complex is incubated with a test sample to obtain an amplification template; the amplification template is subjected to PCR amplification to obtain a sequencing library.

Description

technical field [0001] The present invention relates to the field of biotechnology, more specifically, to an accurate and quantitative ATAC-seq library preparation method and kit. Background technique [0002] At present, we can use the transposase accessible chromosome (ATAC) assay to study open chromatin regions in the genome, which are important binding sites for transcription factors and transcription elements. The methods currently used to study open chromatin regions are: 1. DNase-seq, the naked DNA fragments are cut with DNaseI endonuclease, the cut DNA fragments are sequenced, and compared with the known whole genome sequence, You can find out which part of the cut-off region is, and you can find the developed chromatin region, but this method is cumbersome and takes a long time (2-3 days), and requires a large number of nuclei for experiments (more than one million nuclei), And the repeatability is also poor. 2. ATAC-seq (Assay for Transposase-Accessible Chromatin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C40B50/06C12N15/10
CPCC12Q1/6806C40B50/06C12N15/1093C12Q2525/191C12Q2531/113C12Q2535/122
Inventor 李华俞振勋弓晋欣胥政昊
Owner 苏州京脉生物科技有限公司