Introduction of Chromatin Immunoprecipitation Sequencing (ChIP-Seq)

ChIP (Chromatin Immunoprecipitation) is the cross-linking of intracellular proteins with DNA under physiological conditions and fixing protein-DNA complexes in the living cell state, cutting them randomly into chromatin fragments of a certain length under the action of ultrasound, then precipitating the complex, specifically enriching the target protein-bound DNA fragments, and obtaining information on protein-DNA interactions after DNA purification and its identification.

ChIP-Seq technology, which combines ChIP with high-throughput sequencing, enables efficient and accurate genome-wide screening and identification of DNA binding sites for specific proteins.

Advantages of ChIP-Seq

• Single-base resolution
• No noise caused by DNA fragment hybridization in ChIP microarray
• Wider coverage
• Novel sequences can be discovered

ChIP -Seq Workflow and Protocol

Chromatin Immunoprecipitation Sequencing protocol workflow (Texari L et al. 2021)

1. Sample pretreatment: tissue samples are fixed to cross-link protein-DNA interactions. Formaldehyde is the most commonly used cross-linking agent, and glycine is added to terminate the reaction after cross-linking is complete. Note: The cross-linking agent keeps the complex stable throughout the process, but its action must be reversible so that it can be used for ChIP.

2. Obtain DNA: The cells are lysed to obtain a lysate of whole cells and nuclear DNA is extracted.

3. DNA fragmentation: DNA fragmentation is a key factor in obtaining good ChIP resolution, ideally with fragment sizes between 200 and 1000 bp. Shearing is one of the most difficult steps to control. Shearing can be achieved by sonication and/or nuclease/enzymatic digestion, each with its own advantages and disadvantages. Ultrasonication, although requiring extensive manual manipulation, is well suited for difficult to lyse cells; enzymatic digestion does not require manual manipulation and is suitable for processing large numbers of samples, but its shearing sites are not random.

4. Immunoprecipitation: Antibody immunoprecipitation, where the experimental group is incubated by adding beads pre-conjugated with specific antibodies, resulting in the formation of beads-antibody-target protein-DNA complexes.

5. Uncrosslinking: The antibody-target protein-DNA complex is eluted, the non-specifically bound DNA fragments are removed and uncrosslinked using proteinase K/NaCl treatment, and finally the DNA fragments are purified and recovered.

6. Validation: The ChIP results are validated by qPCR.

7. High-throughput sequencing: DNA samples after ChIP are prepared for ChIP-seq library construction, quality control and sequencing.

ChIP-Seq Data Analysis

Computational analysis in a canonical ChIP-seq analysis (Nakato R and Sakata T 2021)

1. Quality control. Similar to the RNA-seq data analysis process, the raw data obtained from ChIP-seq are first quality checked using fastQC and any unqualified data are further processed.

2. Mapping reads. Compares the sequences of the reads obtained from sequencing to the reference genome by Bowtie, and obtains the alignment files in sam or bam format to obtain the location information of the reads. Samtools software is used to sort the results and remove PCR repeats to obtain the final data for downstream analysis.

3. Peak calling. Using MACS2 to obtain ChIP-seq enrichment information, generating a bed file with detailed location information and confidence for each read.

4. IGV visualization. In order to reduce the size of the bam file and thus reduce the consumption of computer resources during IGV visualization, the bam file needs to be converted into a BigWig file or a TDF format file.

5. Downstream data analysis. Use R-packages Chipseeker for further information mining and visualization of the processed data. GO Annotation by Metascape.

Database of ChIP Sequencing

Applications of ChIP Sequencing

1. ChIP-seq can study histone modifications in order to dissect epigenetic features and biological functions.

Through histone-specific antibodies, histone-DNA complexes with specific modifications are precipitated to obtain histone-bound DNA, and then by sequencing, the distribution of histones on chromosomes can be obtained to identify specific sites related to histone modifications, and the targets of histone-modifying enzymes can also be identified.

2. ChIP-seq can be used to study transcription factor binding sites and resolve the pathway information of the action of the transcription factor.

A transcription factor (TF) is a protein that binds to a specific sequence at the upstream 5' end of a gene and acts as a trans-acting factor, interacting specifically with cis-acting elements of eukaryotic genes, such as promoters and enhancers, to activate or repress gene transcription. ChIP-seq can be used to determine whether a target protein, i.e., a transcription factor, binds to a specific genomic region (e.g., promoter or other DNA binding site).

3. ChIP-seq can be used to map the localization of nucleosomes, which play an important role in a variety of cellular processes such as transcriptional regulation, DNA replication and repair.

4. ChIP-seq technology can study the methylation of DNA, which can cause changes in chromosome structure, DNA conformation, DNA stability and DNA-protein interaction, thus controlling gene expression.

References:

1. Furey T S. ChIP–seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions. Nature Reviews Genetics, 2012, 13(12): 840-852.
2. Texari L, Spann N J, Troutman T D, et al. An optimized protocol for rapid, sensitive and robust on-bead ChIP-seq from primary cells. STAR protocols, 2021, 2(1): 100358.
3. Nakato R, Sakata T. Methods for ChIP-seq analysis: a practical workflow and advanced applications. Methods, 2021, 187: 44-53.

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