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Database establishing method suitable for plant transposase accessible chromatin analysis

A technology of transposase and proximity, applied in the field of molecular biology, can solve the problem of reducing the activity of Tn5 transposase and achieve the effect of reducing interference

Inactive Publication Date: 2018-06-08
奥明(杭州)基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Plant chromatin extracts often contain organelle genomes such as mitochondria and chloroplasts. Since organelle genomes can also be approached and cut by Tn5 transposase, the activity of Tn5 transposase used to digest plant genomes is reduced.

Method used

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  • Database establishing method suitable for plant transposase accessible chromatin analysis
  • Database establishing method suitable for plant transposase accessible chromatin analysis
  • Database establishing method suitable for plant transposase accessible chromatin analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0029] A method for building a library suitable for plant transposase accessible chromatin analysis, comprising the following steps:

[0030] S1: After the plant tissue sample is treated with enzymatic solution TS2 and lysate TS1, cell nucleus suspension A is obtained;

[0031] S2: Staining and sorting the nuclei in the nuclei suspension A to obtain the nuclei B;

[0032] S3: Perform transposition reaction and purification on cell nucleus B to obtain transposition product C;

[0033] S4: performing PCR amplification on the transposition product C to obtain the amplified product D;

[0034] S5: Take part of the amplified product D and perform qPCR to determine the additional required number of PCR cycles, and continue to amplify the remaining amplified product D according to the determined additional required number of PCR cycles to obtain an amplified product E;

[0035]S6: Purify the amplified product E to obtain a single library, and perform fragment sorting on the single ...

Embodiment 1

[0045] A library construction method suitable for the analysis of accessible chromatin of peony flower leaf transposase, refer to figure 1 The flow chart, the specific implementation steps are as follows:

[0046] (1) Separation of nuclei.

[0047] a. Take by weighing 200mg peony flowers and leaves.

[0048] b. Mince the tissue into a homogenate with a sharp blade in a Petri dish placed on ice.

[0049] c. Add 2.5mL TS2 enzymatic hydrolysis solution for 4h to remove the cell wall. The preparation of TS2 enzymolysis solution is: 0.5% cellulase, 0.4% pectinase, 0.3% hemicellulase, 0.5M mannitol, 20mmol / L KCl, 10mmol / L MES (2-(N-morpholine) Ethylsulfonic acid monohydrate, pH 5.7) the mixture was placed in a water bath at 56°C for 10 min, and after cooling to room temperature, 10 mmol / LCaCl was added 2 , 0.1% BSA, and filtered with a 0.45 μm filter.

[0050] d. After enzymatic hydrolysis, centrifuge at 4°C and 400rpm for 10min, and discard the supernatant.

[0051] e. Add 2....

Embodiment 2

[0153] Example 2: A library construction method suitable for tobacco leaf transposase accessible chromatin analysis, refer to figure 1 The flow chart, the specific implementation steps are as follows:

[0154] (1) Separation of nuclei.

[0155] a. Weigh 200 mg of tobacco leaves.

[0156] b. Mince the tissue into a homogenate with a sharp blade in a Petri dish placed on ice.

[0157] c. Add 2.5mL TS2 enzymatic hydrolysis solution for 4h to remove the cell wall. The preparation of TS2 enzymatic hydrolysis solution is: 1.3% cellulase, 0.4% isolated enzyme, 0.3% pectinase, 0.4M mannitol (pH 5.7) mixed solution was placed in 56 ° C water bath for 10 minutes, after cooling to room temperature, 10 mmol / L CaCl 2 , 0.1% BSA, and filtered with a 0.45 μm filter.

[0158] d. After enzymatic hydrolysis, centrifuge at 4°C and 400 rpm for 10 min, and discard the supernatant.

[0159] e. Add 2.5mL pre-cooled TS1 lysate to the precipitate, pipette repeatedly to resuspend, then transfer ...

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Abstract

The invention discloses a database establishing method suitable for plant transposase accessible chromatin analysis. The database establishing method comprises following steps: firstly, plant tender tissue is subjected to pre-treatment, enzymatic hydrolysis is cell walls is carried out, filtering is carried out, DAPI dyeing is adopted to sort and collect nucleuses on a flow cytometer, the obtainednucleuses are filtered so as to reduce interference of cell walls and subcellular organelles on experiment data; Tn5 transposase digestion reaction and purification are carried out, qPCR is adopted to determine circulating number needed by database establishing; at last obtained single library is subjected to equimolar mixing. According to the database establishing method, the high quality on computer library is obtained via optimized two circles of magnetic cell sorting at a certain ratio, so that important experiment method reference is provided for study of scientific research personnel onplant tender tissue accessible transposase nucleus chromatin high flux sequencing.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a library construction method suitable for analyzing plant transposase accessible chromatin. Background technique [0002] ATAC-seq is an innovative epigenetic research technique for the analysis of transposase-accessible chromatin regions by using high-throughput sequencing (Assay for Transposase-Accessible Chromatin with high throughput sequencing). This technology uses transposases to cut open chromatin regions in a certain time and space, and then obtain all the regulatory sequences actively transcribed in the genome under the specific time and space. [0003] Eukaryotic cells assemble genomic DNA and histones into chromatin through different levels of folding, and these precise assembly information play a decisive role in the regulation of gene transcription. The accessibility of chromatin regions is the prerequisite for the interaction between specific trans-acti...

Claims

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Application Information

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IPC IPC(8): C12N15/10C40B50/06
CPCC12N15/1013C40B50/06
Inventor 童云广王华印徐鹭芹赵楠胡浅浅
Owner 奥明(杭州)基因科技有限公司
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