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Construction method of cell Hi-C sequencing library

A construction method and sequencing library technology, applied in chemical libraries, library creation, combinatorial chemistry, etc., can solve the problems of low output of library construction, high cost of library construction, and high output of invalid data, so as to improve the efficiency of library construction Effect

Pending Publication Date: 2020-10-16
天津诺禾致源生物信息科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the existing library construction methods have the following disadvantages: (1) conventional cell Hi-C high-throughput sequencing usually takes 10 7 Building a library for each cell requires a large sample size, which is not suitable for building a library with scarce samples; (2) Due to the method of building a library, the output of invalid data is high, which in turn reduces the amount of data for effective interaction information; (3) The output of a single library construction is low, and high-resolution deep sequencing cannot be performed; (4) The cost of a single library construction is high and the stability is poor. Once the library construction fails, the loss will be large

Method used

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  • Construction method of cell Hi-C sequencing library
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  • Construction method of cell Hi-C sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] 1 cell fixation

[0040] (1) After counting cells, resuspend in 750 μL complete cell culture medium, add 41.7 μL 37% formaldehyde (final concentration 2%), and fix at room temperature for 15 minutes (invert once every 1-2 minutes). Remarks: After cell counting, resuspend in 10ml 1x PBS, add 278μL 37% formaldehyde (final concentration 1%), and fix at room temperature for 10 minutes.

[0041] (2) Add 83.25 μL of 2.5M glycine solution, mix gently, let stand at room temperature for 5 minutes, and then place on ice for 15 minutes. Remarks: Add 555 μL of 2.5M glycine solution, mix gently and let stand at room temperature for 5 minutes, then store on ice.

[0042] (3) Centrifuge the sample solution at 400 g for 5 minutes (4° C.), discard the supernatant.

[0043] (4) Resuspend the cells with 500 μL PBS and transfer to a 1.5 mL centrifuge tube. Centrifuge at 400g for 5 minutes at 4°C, discard the supernatant, repeat the operation once, and collect the fixed cells.

[0044] ...

Embodiment 2

[0134] Utilizing the library construction method under the preferred condition 1 in the present application 1 in Example 1, a Hi-C sequencing library was constructed on duck adipocytes. From Figure 5 and Figure 6It can be seen from the library inspection map of duck adipocytes that the size of the library is concentrated between 400-700bp, which meets the requirements for the distribution of library fragments. The off-machine data of library sequencing was analyzed, and the results are as follows:

[0135] Table 16:

[0136]

[0137] Table 17: Statistics of Duck Adipocyte Sequencing Data

[0138]

[0139] As shown in Table 16 and Table 17, among the data generated from the library constructed by the above 100,000 duck fat cells, the output of invalid data is only 1%, the repetition rate is lower than 20%, and the effective interaction information data is as high as 50%-60% or more.

Embodiment 3

[0141] The comparison of library data between the method of the present invention (100,000 cells) and the conventional cell Hi-C method (5,000,000 cells) is shown in Table 18:

[0142] Table 18:

[0143]

[0144] As shown in Table 18, the Hi-C library data of 100,000 cells using the method of the present invention compared with the library data constructed by the conventional Hi-C method of 5 million cells, the effective interaction information The data reaches more than 60%, and the proportion of invalid data is lower.

[0145] In order to further obtain a better effect of cross-linking and fixation treatment, the inventors also screened the cross-linking and fixation method for cells with a low initial amount, see the following examples for details.

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Abstract

The invention provides a construction method of a cell Hi-C sequencing library. The construction method comprises the following steps: carrying out formaldehyde cross-linking immobilization on 10<5>-order cells by adopting a cross-linking system of which the volume is less than or equal to 800 muL and the final concentration of formaldehyde is 1.5-2.2%, and then treating the cross-linked cells byadopting an optimized cracking system and temperature to obtain a cell nucleus suspension; carrying out Hi-C library construction on the cell nucleus to obtain an Hi-C sequencing library. The cell membrane lysis solution comprises the following components with final concentrations: Tris-HCl with the pH value of 7.8 to 8.2 and the concentration of 9 to 11mM, NaCl with the concentration of 9 to 11mM, CA-630 with the concentration of 0.1 to 0.3 percent and a protease inhibitor with the concentration of 1*. A small-volume system fixes the initial quantity of low cells, optimizes a cell membrane lysis system and reaction conditions, ensures the integrity of cell nucleuses, and improves the quantity and purity of DNA of the cell nucleuses, thereby improving the library building efficiency (improving the proportion of effective data).

Description

technical field [0001] The present invention relates to the field of sequencing library construction, in particular to a method for constructing a cellular Hi-C sequencing library. Background technique [0002] Chromosome Conformation Capture (3C) is a useful tool for studying the spatial interaction of the three-dimensional genome structure. In recent years, the Hi-C technology extended by 3C, 4C, 5C and other technologies combined with high-throughput sequencing methods can obtain the three-dimensional structure of the whole genome. Structural interaction information, such as euchromatin and heterochromatin (A / B compartment), topological domain (TAD), and chromatin loops (Loops), etc. The spatial structure of chromatin has always been a research hotspot, and the interaction between genes and remote elements plays an important role in the regulation of gene expression and other genomic activities. Dense matrices of chromatin interactions can be used to determine the spatia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06
CPCC40B50/06
Inventor 李新刘贝贝李瑞强赵桂仿
Owner 天津诺禾致源生物信息科技有限公司
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