CN117070613A - Method for capturing interaction of DNA protein of very small amount of cells - Google Patents

Abstract

The invention provides a method for capturing interaction of a very small amount of cell DNA protein, which comprises the following steps: the pA/G-Tn5 capable of being specifically combined with the antibody is pre-incubated, cells are lysed through cell lysate, a DNA sequence combined with the target protein is identified and sheared by means of a complex of the Tn5 and the specific antibody, and finally the obtained fragment is subjected to library construction to obtain a library which can be used for second-generation sequencing. The method can simultaneously obtain high-quality DNA protein interaction data in a whole genome range, is particularly suitable for research samples with scarce quantity such as embryos, cancers, immune cells and the like, and provides possibility for understanding protein DNA interaction under a small quantity of cells.

Description

Method for capturing interaction of DNA protein of very small amount of cells

Technical Field

The invention relates to a DNA protein interaction capturing technology and a second generation sequencing standard sample library construction method.

Background

In recent years, with the development of second generation sequencing technology, more technology has emerged to help people understand each event in biology. And detecting the interaction of DNA and protein in the whole genome of the cell nucleus can help us understand the mechanism of the trans-regulatory elements and cis-regulatory elements in aspects of gene expression regulation and the like. In recent years, conventional ChIP-seq technology has been further optimized for the purpose of detecting histone modifications in hundreds of cells. By means of the method, three research groups in 2016 independently report dynamic changes of histone H3K4me3 and H3K27me3 modification in the early embryo development process of mice, and important basis and reference are provided for researching epigenetic dynamics and mechanism in the early embryo development process, so that development of related fields is promoted.

However, unlike the detection of histone modifications, the binding of important proteins to DNA, such as RNA polymerase II, CTCF, multiple transcription factors, etc., does not have a pattern of binding closely to DNA as with histone in the form of nucleosomes, which makes us unable to apply the optimized ChIP-seq, even if one already has the ability to detect histone modifications with high sensitivity on the order of as low as hundred, for experimental purposes of detecting the interaction of RNA polymerase II and other transcription factors with DNA.

In view of the above facts and difficulties encountered, the objective of detecting the distribution of RNA polymerase II across the whole genome in small numbers of cells was achieved by developing new methods, rather than optimizing the existing ChIP-seq method.

In fact, previous studies have shown that strategies to detect interactions of DNA and proteins across the genome, in addition to traditional ChIP approaches, are ChIC (chromatin immuno cleavage) and ChEC (chromatin endogenous cleavage) strategies. Unlike the antibody co-precipitation-based method in ChIP, the basic principle of ChEC/ChEC is based on bringing the nuclease bound to protein a/G to the binding site of the target protein and DNA recognized by the antibody through the interaction of the antibody with the protein a/G, achieving in situ cleavage, and identifying sequence information of DNA bound to the target protein through downstream enrichment and amplification.

In recent years, transposase Tn5 from prokaryotic cells has been widely used in DNA fragmentation and DNA library construction in experimental methods related to molecular biology and sequencing. Because of the characteristics of the transposase, tn5 can realize that the self-contained linker sequence is connected to the target DNA fragment while DNA is cut, and the excellent shearing, namely connection, performance of the method is more stable and efficient than the traditional method that the process of cutting DNA and then connecting the linker sequence through DNA ligase, so that the method is favored. A number of widely used sequencing-related methods employ Tn5 pooling, such as SMART-seq2, chIA-PET, hi-C, and ATAC-seq. Considering the excellent characteristics of Tn5 and combining with our experimental purposes, we hypothesize that integrating Tn5 with the ChIC strategy can effectively improve the sensitivity of the ChIC strategy and establish a technology for capturing interaction of a very small amount of cell DNA proteins based on Tn5 and ChIC.

Disclosure of Invention

The invention mainly solves the problem of the interaction capturing technology for the DNA protein of the very small amount of cells. The method can detect whole gene DNA protein interactions with initial cell amounts as low as several tens. The experimental steps and the key tool enzyme Tn5 involved in the experiment are brand new design and development.

The invention provides a DNA protein interaction capturing and second-generation sequencing standard sample library construction method. According to an embodiment of the invention, the method comprises:

subjecting the cell lysis buffer to a lysis treatment so as to obtain a cell lysis solution;

co-incubating pA/G-Tn5 with the antibody to obtain a Tn5 antibody complex;

adding the Tn5 antibody complex into a cell lysate, adding magnesium ions, heating, and shearing DNA bound by a target protein by using Tn 5;

the library was obtained by PCR using the DNA product and sequenced.

The traditional DNA protein interaction capturing technology experiment has long method period and low efficiency due to complicated steps. To date, conventional methods still require millions of initial cell amounts to complete an experiment. In the method, after improvement and simplification of each step, the technology can use a very small number of cells (as low as hundreds) to obtain high-quality whole genome DNA protein interaction data within 1 day, thereby providing possibility for revealing the regulation of the transcription level of a small number of cells. As shown in FIG. 1, the number of cells required for the present method is significantly lower than for all methods (500) compared to other published articles (millions of cells are also required at a minimum). As shown in FIG. 2, our method is able to effectively monitor the distribution of RNA polymerase II across the genome at a level as low as 500 cells. The final modified process has good reproducibility (fig. 3). Thus, in summary, our very small amount of DNA protein interaction capture technology can use hundreds of experimental data that would have been obtained with millions of cells. Provides a feasible experimental scheme for more experiments which can not provide a large number of cells.

According to an embodiment of the invention, the number of the cells is 500 to 1x10 ⁶ Preferably 500.

According to an embodiment of the invention, the lysis buffer volume used for performing the cell lysis treatment is 6. Mu.L.

According to an embodiment of the invention, the lysis buffer comprises: 10mM Tris-HCl, ph=7.4; 150mM NaCl;0.5mM Sperminine; 1x Roche complex.

According to an embodiment of the present invention, the time of the first lysis treatment is 5 to 15 minutes.

According to an embodiment of the invention, the DNA protein-interacting DNA library is obtained by:

lysing the cells to obtain a cell lysate;

incubating pA/G-Tn5 with the antibody to obtain a Tn5 antibody complex;

adding the Tn5 antibody complex into a cell lysate, and performing enzyme digestion and PCR to obtain a DNA library;

wherein the cycle number in the pre-amplification procedure is 8-20.

According to an embodiment of the invention, the protein DNA interaction DNA library is obtained by:

(1) Lysing cells using the lysate, adding the antibody Tn5 complex;

(2) Cleaving the DNA bound by the protein of interest through the antibody Tn5 complex;

(3) Specific sequences of the protein-binding DNA library were obtained by PCR and second generation sequencing.

In another aspect of the invention, the invention provides a method for detecting DNA protein interactions in a small number of cells. According to an embodiment of the invention, the method comprises:

(1) The following reagents were provided:

a lysis buffer comprising: 10mM Tris-HCl, ph=7.4; 150mM NaCl;0.5mM Sperminine; 1x Roche complex;

absolute ethyl alcohol

Ultrapure water

protein A/G-Tn5 fusion protein (pA-Tn 5 or pG-Tn 5)

AMPure beads

Qubit

Phenol chloroform

Carrier RNA (carrier RNA)

Sodium acetate (3M)

Glycogen (glycogen)

5x TTBL(Vazyme，TD502)

PCR kit (Vazyme, TD 601)

Digitonin

PCR primer kit (Vazyme, TD 202)

Incorporated internal reference DNA (spike-in DNA, drosophila genomic DNA sheared by Tn5, 0.025 pg/ul)

The specific process comprises the following steps:

cells or embryos are collected and counted in a break-up, generally, the number of cells should be between 200 and 1 million, depending on the abundance of the protein of interest and the antibody properties.

The antibody was pre-incubated with pA-Tn5 or pG-Tn 5. The choice of Protein A, G depends largely on the type of antibody used. In this study, pol II selected pG-Tn5 and H3K4me3 and H3K27ac selected pA-Tn5. The completely dissolved 5% digitonin was added to 1ml Buffer 1 and thoroughly mixed to prepare D-B1. To a low adsorption of 200ul EP tube was added 7ul DB-1,0.5ul pA/G-Tn5 and 0.5ug antibody, and after mixing, placed at 4℃for 30 minutes.

To the collected cells or embryos, 6ul of DB-1 was added to lyse the cells, and the cells were left at 4℃for 10 minutes.

35ul DB-1, pre-prepared antibody, pA/G-Tn5 complex and 12.5ul5xTTBL buffer were added to the lysed cells and placed on a horizontal mixer at 37℃400rpm for 30 min.

2ul of vector RNA,2ul of reference DNA and 65ul of TE buffer were added. The solution was transferred to a Phase-lock (Tiangen) tube. 130ul of phenol chloroform was added thereto to extract and purify the DNA. The supernatant was then taken, added with 650ul absolute ethanol, 24ul sodium acetate and 2ul glycogen, and placed in a-20℃refrigerator overnight.

After centrifugation at low temperature of 4℃for 30 minutes, a precipitate was left, washed once with 80% ethanol, dissolved in 29ul of water, and then PCR was performed with PCR reagents in TD502 (Vazyme), 500 cells corresponding to 16 PCR cycles.

Qubit detects DNA concentration, 2100 nuclear analyzer detects fragment size, followed by Hi-Seq 2500 or Xten sequencing.

Bioinformatics analysis.

The method can simultaneously realize the construction of the DNA sequencing library for interaction of a small amount of cell DNA proteins in a short time (1 day), thereby providing possibility for understanding interaction and cooperative regulation of protein DNA genome level.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 shows the effect of the method compared to two replicates of the method at different cell numbers;

FIG. 2 shows the capture rate of the method, showing the effect of the method compared to conventional methods at different cell numbers;

the reproducibility of the method of fig. 3 is shown, demonstrating the reproducibility of the method at different cell numbers.

Detailed Description

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.

Example 1

1. Reagent preparation

Buffer 1 (Buffer 1)

10mM Tris-HCl

150mM NaCl

0.5mM Spermidine

1x Roche complete

Other reagents

Absolute ethyl alcohol

Ultrapure water

protein A/G-Tn5 fusion protein (pA-Tn 5 or pG-Tn 5)

AMPure beads

Qubit

Phenol chloroform

Carrier RNA (carrier RNA)

Sodium acetate (3M)

Glycogen (glycogen)

5x TTBL(Vazyme，TD502)

PCR kit (Vazyme, TD 601)

Digitonin

PCR primer kit (Vazyme, TD 202)

Incorporated internal reference DNA (spike-in DNA, drosophila genomic DNA sheared by Tn5, 0.025 pg/ul)

2. Method specific flow

1. Cells or embryos are collected and counted in a break-up, generally, the number of cells should be between 200 and 1 million, depending on the abundance of the protein of interest and the antibody properties.

2. The antibody was pre-incubated with pA-Tn5 or pG-Tn 5. The choice of Protein A, G depends largely on the type of antibody used. In this study, pol II selected pG-Tn5 and H3K4me3 and H3K27ac selected pA-Tn5. The completely dissolved 5% digitonin was added to 1ml Buffer 1 and thoroughly mixed to prepare D-B1. To a low adsorption of 200ul EP tube was added 7ul DB-1,0.5ul pA/G-Tn5 and 0.5ug antibody, and after mixing, placed at 4℃for 30 minutes.

3. To the collected cells or embryos, 6ul of DB-1 was added to lyse the cells, and the cells were left at 4℃for 10 minutes.

4. 35ul DB-1, pre-prepared antibody, pA/G-Tn5 complex and 12.5ul5xTTBL buffer were added to the lysed cells and placed on a horizontal mixer at 37℃400rpm for 30 min.

5. 2ul of vector RNA,2ul of reference DNA and 65ul of TE buffer were added. The solution was transferred to a Phase-lock (Tiangen) tube. 130ul of phenol chloroform was added thereto to extract and purify the DNA. The supernatant was then taken, added with 650ul absolute ethanol, 24ul sodium acetate and 2ul glycogen, and placed in a-20℃refrigerator overnight.

After centrifugation at 6.4℃for 30 min with a low temperature centrifuge, a precipitate was left, washed once with 80% ethanol, dissolved in 29ul of water, and then PCR was performed with PCR reagents in TD502 (Vazyme), 500 cells corresponding to 16 PCR cycles.

Qubit detection DNA concentration, 2100 nuclear analyzer detection fragment size, followed by Hi-Seq 2500 or Xten sequencing.

8. Bioinformatics analysis.

As shown in FIG. 1, the number of cells required for the present method is significantly lower than for all methods (500) compared to other published articles (millions of cells are also required at a minimum). As shown in FIG. 2, our method is able to effectively monitor the distribution of RNA polymerase II across the genome at a level as low as 500 cells. The final modified process has good reproducibility (fig. 3). Thus, in summary, our very small amount of DNA protein interaction capture technology can use hundreds of experimental data that would have been obtained with millions of cells. Provides a feasible experimental scheme for more experiments which can not provide a large number of cells.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (5)

1. A method for constructing a library of DNA protein interaction capture and second generation sequencing standards, comprising:

subjecting the cell lysis buffer to a lysis treatment so as to obtain a cell lysis solution;

co-incubating pA/G-Tn5 with the antibody to obtain a Tn5 antibody complex;

adding the Tn5 antibody complex into a cell lysate, adding magnesium ions, heating, and shearing DNA bound by a target protein by using Tn 5;

the library was obtained by PCR using the DNA product and sequenced.

2. The method according to claim 1, wherein the number of cells is 500 to 1x10 ⁶ Preferably 500.

3. The method of claim 1, wherein the cell lysis treatment is performed with a lysis buffer volume of 6 μl;

optionally, the lysis buffer comprises: 10mM Tris-HCl, ph=7.4; 150mM NaCl;0.5mM Sperminine; 1x Roche complex;

optionally, the first lysis treatment is for a period of time of 5 to 15 minutes.

4. The method of claim 1, wherein the DNA protein-interacting DNA library is obtained by:

lysing the cells to obtain a cell lysate;

incubating pA/G-Tn5 with the antibody to obtain a Tn5 antibody complex;

adding the Tn5 antibody complex into a cell lysate, and performing enzyme digestion and PCR to obtain a DNA library;

wherein the cycle number in the pre-amplification procedure is 8-20.

5. The method of claim 1, wherein the protein DNA interactive DNA library is obtained by:

(1) Lysing cells using the lysate, adding the antibody Tn5 complex;

(2) Cleaving the DNA bound by the protein of interest through the antibody Tn5 complex;

(3) Specific sequences of the protein-binding DNA library were obtained by PCR and second generation sequencing.