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Chromatin immunoprecipitation sequencing steps

Chromatin immunoprecipitation sequencing (ChIP-seq) is an important technology to study gene expression regulation, DNA modification and protein-DNA interaction. Through this technology, researchers can efficiently analyze protein-DNA binding sites on chromatin, further revealing the regulatory mechanism of gene expression.

Chromatin immunoprecipitation sequencing

1. Cell treatment and cross-linking1. Cell culture: Culture the target cells to the appropriate density and state to ensure that the cells are in an active growth state. 2. Formaldehyde cross-linking: Use cross-linking agents such as formaldehyde to treat cells to form a stable cross-linking between proteins and DNA in the cells. The formaldehyde concentration and treatment time need to be optimized according to experimental needs.

2. Cell lysis and chromatin fragmentation1. Cell lysis: Use appropriate lysis buffer to lyse the cells and release chromatin in the cell nucleus. 2. Chromatin fragmentation: Chromatin fragmentation is fragmented into DNA fragments of appropriate size by sonication or enzymatic decomposition, usually with a length of 100~500bp. This step facilitates subsequent immunoprecipitation and sequencing.

3. Immunoprecipitation1. Antibody incubation: Add specific antibodies to the cell lysate, and the antibodies will bind to the target protein. 2. Immune complex precipitation: Use protein A/G magnetic beads or other methods to precipitate the antibody-protein-DNA complex. Unspecifically bound DNA and protein were removed by washing the magnetic beads.

IV. DNA purification1. Decrosslinking: Use high temperature and protease K to treat the immunoprecipitation complex to unblock the crosslinking between protein and DNA, and release the purified DNA fragments. 2. DNA extraction: Purified DNA fragments were extracted by centrifugation, filtration, etc. for subsequent sequencing library construction.

5. Sequencing library construction1. DNA end repair: Perform end repair of purified DNA fragments to have a sticky end suitable for sequencing. 2. Add a sequencing linker: Add a sequencing linker at the end of the DNA fragment, which will be used for subsequent sequencing reactions. 3. Library amplification: The sequencing library is amplified by PCR and other methods to obtain a sufficient number of DNA fragments for sequencing.

6. High-throughput sequencing1. Sequencing platform selection: Select a suitable sequencing platform according to experimental needs, such as Illumina, etc. 2. Sequencing reaction: Add the sequencing library to the sequencer for high-throughput sequencing. Sequencing will generate a large amount of DNA sequence data for subsequent bioinformatics analysis.

7. Data Analysis1. Data Preprocessing: Preprocessing steps such as quality control and removing low-quality sequences and linker sequences on the sequencing data. 2. Alignment and annotation: Aligate the pretreated sequences onto the reference genome to determine the site where proteins interact with DNA. At the same time, gene annotation and functional analysis were performed on these loci. 3. Peak detection and analysis: Use specialized software tools to detect ChIP-Seq signal peaks, which represent the binding sites of proteins on the genome. The relationship between these peaks and gene expression, epigenetic markers, etc. was further analyzed. 4. Difference analysis: For multi-sample experiments, differential analysis can be performed to identify the differential sites of protein and DNA interaction between different samples.

Chromatin immunoprecipitation sequencing (ChIP-Seq) is a complex and fine experimental technique involving multiple critical steps and fine operations. Each step requires strict control of experimental conditions and quality to ensure the accuracy and reliability of the results.

Chromatin immunoprecipitation sequencing steps

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