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Rapid library building method for identifying target protein chromatin binding map

A technology for chromatin and target identification, which is applied in the field of rapid library building for identifying chromatin binding maps of target proteins.

Active Publication Date: 2021-03-26
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technique still has defects such as long operation time and large loss during the operation, which limits the development and application of this technique on precious samples and single cells.

Method used

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  • Rapid library building method for identifying target protein chromatin binding map
  • Rapid library building method for identifying target protein chromatin binding map
  • Rapid library building method for identifying target protein chromatin binding map

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090]Example 1: The effect of secondary antibody and recombinant fusion transposase Protein A-TN5 and Protein Ag-TN5 pre-bonded to library yield.

[0091]Process diagramFigure 7 The specific process is as follows:

[0092]1. Collect cells. Take about 100,000 293FT cells, and the cells were centrifuged at room temperature for 600 x g. It was cleaned twice with a cleaning buffer (20 mm hepes (pH 7.5), 150 mm NaCl, 0.5 mm, 1x protease inhibitorscocktail). The cells were resuspended in 90 μL of cleaning buffer.

[0093]2. Activate magnetic beads and capture cells. 10 μl of CONA magnetic beads were added to the binding buffer (20 mM HEPES (pH 7.5), 10 mM KCl, 1 mM CaCl2, 1 mm MnCl2) washed twice. The magnetic beads were hung in 10 μl binding buffer. The magnetic beads were added to the cells, and the room temperature was 10 to 10 min.

[0094]3. Anti-combination. Three kinds of dilutions were diluted in 1: 100, using an anti-dilution buffer (20 mm hepes (pH 7.5), 150 mm NaCl, 0.5 mm amine, 0.05% Di...

Embodiment 2

[0109]Example 2: The effect of different genomic DNA extraction methods on library yield.

[0110]Using recombinant fusion transposase Protein Ag-TN5 and H3K27ME3 antibody to verify the effect of different genomic DNA extraction methods on library production, flow schematicFigure 12 The specific process is as follows:

[0111]1. Collect cells. Take about 100,000 293FT cells, and the cells were centrifuged at room temperature for 600 x g. It was cleaned twice with a cleaning buffer (20 mm hepes (pH 7.5), 150 mm NaCl, 0.5 mm, 1x protease inhibitorscocktail). The cells were resuspended in 90 μL of cleaning buffer.

[0112]2. Activate magnetic beads and capture cells. 10 μl of CONA magnetic beads were added to the binding buffer (20 mM HEPES (pH 7.5), 10 mM KCl, 1 mM CaCl2, 1 mm MnCl2) washed twice. The magnetic beads were hung in 10 μl binding buffer. The magnetic beads were added to the cells, and the room temperature was 10 to 10 min.

[0113]3. Anti-combination. Two kinds of dilutions were dilu...

Embodiment 3

[0133]Example 3: Protein A-TN5 and Protein AG-TN5 in FTCT-SEQ buildings.

[0134]According to the improvement of Examples 1 and Example 2, we invented a rapid settlement method for identifying a target protein DNA binding map and named FTCT-SEQ. The reorganized fusion transposase Protein A-TN5 and Protein AG-TN5 verify the construction of the method of the process of the invention, the specific process is as follows:

[0135]1. Collect cells. Take about 100,000 293FT cells, and the cells were centrifuged at room temperature for 600 x g. It was cleaned twice with a cleaning buffer (20 mm hepes (pH 7.5), 150 mm NaCl, 0.5 mm, 1x protease inhibitorscocktail). The cells were resuspended in 90 μL of cleaning buffer.

[0136]2. Activate magnetic beads and capture cells. 10 μl of CONA magnetic beads were added to the binding buffer (20 mM HEPES (pH 7.5), 10 mM KCl, 1 mM CaCl2, 1 mm MnCl2) washed twice. The magnetic beads were hung in 10 μl binding buffer. The magnetic beads were added to the cells, ...

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Abstract

The invention discloses a rapid library building method for identifying a target protein chromatin binding map, which comprises the following steps: collecting sample cells, adding activated magneticbeads into the cells, incubating, and incubating the magnetic beads with a primary antibody; pre-binding a secondary antibody with recombinant fusion transposase; incubating the magnetic beads with the pre-combined secondary antibody and recombinant fusion transposase, adding a transposase activator and a cell perforating agent into the magnetic beads, and incubating; and terminating the transposase reaction, and carrying out library amplification. According to the method, the process of a traditional method is remarkably simplified, the library building time is shortened, the library buildingefficiency is improved, the loss is small, and the requirement for library building materials is lower. Meanwhile, three DNA standard substances with different concentrations are doped into the library building process, so that the effect of accurately quantifying the DNA copy number in the library can be achieved. The method is very suitable for researching a small amount of precious samples orsamples with different treatment states.

Description

Technical field[0001]The present invention relates to a rapid construction library method for identifying a target protein-stained color binding, which belongs to the field of biotechnology.Background technique[0002]Chromatin Immunoprecipitation Assay (Chip) is a key means to study gene expression regulation, DNA damage repair and apparent genetic modification, and an important tool for exploring the cause of life processes and cellopathy. With the rise and development of high-throughput sequencing techniques, chromatin immunocolite sequencing techniques (CHIP-SEQ) becomes a high-efficiency tool for studying DNA binding proteins in chromatin. As of the current location, Encyclopedia of Dna Elements, ENCODE and Epigenomics Projects have accommodated chromatin binding maps of more than 200 DNA binding proteins, including applary Genetic modification and its regulatory protein, transcriptional regulatory factor, chromatin structure regulatory protein, etc. In recent years, increasing d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06
CPCC40B50/06
Inventor 江翱曹振宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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