Unlock instant, AI-driven research and patent intelligence for your innovation.

A library construction method for analysis of chromatin accessibility of transposases in tissue lymphocytes

A technology of lymphocytes and transposases, applied in the field of molecular biology, can solve problems such as time urgency and unconditional cell culture, and achieve the effect of reducing background interference and cost

Active Publication Date: 2022-01-18
奥明(杭州)基因科技有限公司
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ATAC-seq is now commonly used to systematically identify cis-regulatory regions and DNA footprints in animal genomes. At present, this method has gradually become popular in cultured single-cell suspensions, but direct use of tissues for lymphocytes The detection experiment has not been reported yet. First, the time period is greatly saved, and there is no need to digest the tissue for culture first. Second, some scientific research units do not have the conditions to carry out cell culture, and the time is tight and they want to directly use the collected animal tissues for ATAC-seq research. in accordance with

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A library construction method for analysis of chromatin accessibility of transposases in tissue lymphocytes
  • A library construction method for analysis of chromatin accessibility of transposases in tissue lymphocytes
  • A library construction method for analysis of chromatin accessibility of transposases in tissue lymphocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0030] A method for building a library for analyzing the accessible chromatin of lymphocyte transposases, comprising the following steps:

[0031] S1: After the animal tissue is shredded and filtered, the single cell suspension A is obtained;

[0032] S2: Separating mononuclear cells from the mononuclear suspension A to obtain a mononuclear cell solution B;

[0033] S3: performing a cell lysis transposition reaction and purification on the mononuclear cell liquid B to obtain a transposition product C;

[0034] S4: performing PCR amplification on the transposition product C to obtain the amplified product D;

[0035] 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;

[0036] S6: Purify the amplified product E to obtain a single library, and perform fra...

Embodiment 1

[0044] A library construction method suitable for the analysis of mouse tissue lymphatic transposase accessibility chromatin, refer to figure 1 The flow chart, the specific implementation steps are as follows:

[0045] (1) Preparation of mononuclear cell suspension

[0046] a. Cell separation solution preparation: Iodixanol 16.0%, RPMI-1640 medium 73.0%, ultrapure water 11%, density: 1.088±0.002g / mL;

[0047] b. Weigh 20 mg of mouse spleen tissue;

[0048] c. Use ophthalmic scissors to cut the mouse tissue into a homogenate in 5mL of separation solution;

[0049] d. Immediately transfer the separation solution with spleen cells suspended to a 15mL centrifuge tube, and cover with 500uL RPMI1640 medium (keep the boundary of the liquid level clear);

[0050] e. Carry out centrifugation, 4°C, centrifugal force 800×g, centrifugation time 30min, absorb the lymphocyte layer, located below the covering layer RPMI1640 medium, above the separation liquid;

[0051] (2) Counting, wash...

Embodiment 2

[0144] A library construction method suitable for the analysis of transposase accessible chromatin in rat tumor-infiltrating tissue lymphocytes, refer to figure 1 The flow chart, the specific implementation steps are as follows:

[0145] (1) Preparation of mononuclear cell suspension

[0146] a. Cell separation solution preparation: Iodixanol 15.6%, RPMI-1640 medium 74.4%, ultrapure water 10%, density: 1.084±0.002g / mL;

[0147] b. Weigh 20 mg of rat tumor infiltration tissue;

[0148] c. Use ophthalmic scissors to cut the tumor tissue into a homogenate shape in 5mL of separation solution;

[0149] d. Immediately transfer the separation solution suspended with tumor-infiltrating tissue cells to a 15mL centrifuge tube, and cover with 500uLRPMI1640 medium (keep the boundary of the liquid surface clear);

[0150] e. Carry out centrifugation, the centrifugal force is 800×g, and the centrifugation time is 30 minutes, and the lymphocyte layer is sucked, which is located under the ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for building a library for the analysis of the accessible chromatin of tissue lymphocyte transposase. The invention first pre-treats the animal tissue, shreds it and filters it, and uses the cell separation liquid to separate mononuclear cells to ensure collection. The mononuclear cells have high cell activity. Add DNaseI to digest the DNA of dead cells, and use cell washing solution to wash, effectively reduce background interference and cost, then perform Tn5 transposase digestion reaction and purification, and qPCR to determine the cycle required for library construction Finally, equimolar mixing was performed on the single library obtained, and the optimized two-round magnetic bead ratio sorting was used to obtain a high-quality library on the machine, which is easy for researchers to study animal tissue lymphocytes. Throughput sequencing provides an important experimental method reference.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for building a library for analyzing the accessible chromatin of a tissue lymphocyte transposase. Background technique [0002] Lymphocytes play a very important role in the process of immune response. Its number and functional state reflect the immune state of the body, which is related to some congenital or acquired immunodeficiency diseases, infectious diseases and malignant tumors and other immune dysfunction and other diseases are closely related. Therefore, the study of lymphocytes is of great significance to the diagnosis, prevention and treatment of various diseases. [0003] 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 chr...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12N15/10
CPCC12N15/1013C40B50/06C12Q2531/113
Inventor 童云广徐鹭芹
Owner 奥明(杭州)基因科技有限公司