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A Rapid Library Construction Method for Identifying Chromatin Binding Profiles of Target Proteins

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

Active Publication Date: 2021-05-11
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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  • A Rapid Library Construction Method for Identifying Chromatin Binding Profiles of Target Proteins
  • A Rapid Library Construction Method for Identifying Chromatin Binding Profiles of Target Proteins
  • A Rapid Library Construction Method for Identifying Chromatin Binding Profiles of Target Proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1: Effect of pre-combination of secondary antibody with recombinant fusion transposase Protein A-Tn5 and Protein AG-Tn5 on library yield.

[0091] See flow chart Figure 7 , the specific process is as follows:

[0092] 1. Harvest the cells. Take about 100,000 293FT cells and centrifuge at 600xg for 5 min at room temperature to collect the cells. Wash 2 times with wash buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1x Protease inhibitorscocktail). Resuspend the cells in 90 µL of wash buffer.

[0093] 2. Activate magnetic beads and capture cells. Take 10 μL ConA magnetic beads, add binding buffer (20 mM HEPES (pH7.5), 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) to wash the magnetic beads twice. Resuspend the magnetic beads in 10 µL of binding buffer. Add the magnetic beads to the cells and incubate with rotation at room temperature for 5-10 min.

[0094] 3. Primary antibody binding. Dilute the three kinds of antibodies at 1:100 with primary antibody ...

Embodiment 2

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

[0110] Use the recombinant fusion transposase Protein AG-Tn5 and H3K27me3 antibody to verify the effect of different genomic DNA extraction methods on the library yield. The flow chart is shown in Figure 12 , the specific process is as follows:

[0111] 1. Harvest the cells. Take about 100,000 293FT cells and centrifuge at 600xg for 5 min at room temperature to collect the cells. Wash 2 times with wash buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1x Protease inhibitorscocktail). Resuspend the cells in 90 µL of wash buffer.

[0112] 2. Activate magnetic beads and capture cells. Take 10 μL ConA magnetic beads, add binding buffer (20 mM HEPES (pH7.5), 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) to wash the magnetic beads twice. Resuspend the magnetic beads in 10 µL of binding buffer. Add the magnetic beads to the cells and incubate with rotation at room temperature for 5-10 mi...

Embodiment 3

[0133] Example 3: Library construction effect of Protein A-Tn5 and Protein AG-Tn5 in FTCT-seq.

[0134] According to the improvement of Example 1 and Example 2, we invented a rapid library construction method for identifying the DNA binding profile of the target protein, and named it FTCT-seq. And use recombinant fusion transposase Protein A-Tn5 and Protein AG-Tn5 to verify the effect of building the library of the method process of the present invention, specific process is as follows:

[0135] 1. Harvest the cells. Take about 100,000 293FT cells and centrifuge at 600xg for 5 min at room temperature to collect the cells. Wash 2 times with wash buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1x Protease inhibitorscocktail). Resuspend the cells in 90 µL of wash buffer.

[0136] 2. Activate magnetic beads and capture cells. Take 10 μL ConA magnetic beads, add binding buffer (20 mM HEPES (pH7.5), 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) to wash the magnetic beads twic...

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Abstract

The invention discloses a method for quickly building a library for identifying the chromatin binding map of a target protein. The steps include: collecting sample cells, adding activated magnetic beads into the cells for incubation, and incubating the magnetic beads with a primary antibody; secondary antibody and recombinant Pre-binding of fusion transposase; incubation of magnetic beads with pre-bound secondary antibody and recombinant fusion transposase. Add transposase activator and cell perforation agent to the magnetic beads and incubate; terminate the transposase reaction and perform library amplification. The invention significantly simplifies the flow of the traditional method, shortens the time for building a library, improves the efficiency of building a library, has small losses, and has lower requirements on materials for building a library. At the same time, by incorporating three DNA standards of different concentrations into the library construction process, the effect of accurately quantifying the DNA copy number in the library can be achieved. This method is very suitable for the study of small, precious samples or samples in different processing states.

Description

technical field [0001] The invention relates to a rapid library building method for identifying target protein chromatin binding patterns, and belongs to the field of biotechnology. Background technique [0002] Chromatin immunoprecipitation assay (ChIP) is a key method to study gene expression regulation, DNA damage repair and epigenetic modification, and it is also an important tool for people to explore the cause of life process and cell disease. With the rise and development of high-throughput sequencing technology, chromatin immunoprecipitation sequencing technology (ChIP-seq) has become an efficient tool for people to study the binding map of DNA-binding proteins on chromatin. As of now, the Encyclopedia of dna elements (ENCODE) and the Roadmap Epigenomics Project (Roadmap Epigenomics Project) have collected the chromatin binding maps of more than 200 DNA binding proteins, including epigenetic Genetic modification and its regulatory proteins, transcriptional regulator...

Claims

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Application Information

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Patent Type & Authority Patents(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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