ChIP Sequencing

ChIP-Seq, short for Chromatin immunoprecipitation sequencing, is a next-generation based sequencing method to comprehensively study the interaction between proteins and DNA. It takes advantage of the specificity of the antigenic antibody response to provide a fidelity reflection of the binding of protein factors to genomic DNA in vivo.

Overview

In recent years, due to the continuous development and refinement of ChIP-Seq its application has evolved from the study of interactions between target proteins and known target sequences to the study of interactions between target proteins and unknown sequences throughout the genome; from studying the interaction of a target protein with DNA to the interaction of two proteins co-associated with DNA; from studying the modification of histones in promoter regions to protein complexes bound to DNA sequences. And ChIP-Seq can be widely used in transcription factor binding sites (TFBSs) and histone modification studies. This technique uses reagents such as formaldehyde to cross-link and immobilize protein-DNA complexes in cells, lyses and ultrasonic processing cells, and immunoprecipitates them with antibodies specific to the target protein (or labeled antibodies) to obtain the target protein-DNA complexes. High-throughput sequencing of the DNA in the cross-linked isolated complexes allows the identification of DNA sequences that may interact with the target protein.

Genome-wide mapping of protein-DNA interactions and epigenetic marks is essential for a comprehensive understanding of transcriptional regulation in organisms. DNA-binding proteins, including transcription factors, epigenetic factors, and chromatin modifiers, control gene expression in the organism. Localizing the binding sites of DNA-binding proteins in the genome is essential for decoding gene regulatory networks.

By performing RNA-Seq after overexpression/knockout/disruption of a specific gene to search for significantly up- or down-regulated downstream genes affected by the gene; ChIP-Seq can search for genetic information of DNA binding sites where histones, transcription factors, etc. interact. When RNA differentially expressed genes are analyzed in conjunction with ChIP-Seq technology, it helps us to explore the mechanisms by which transcription factors, cofactors and histone modifications regulate gene expression.

Project Workflow

Bioinformatics Analysis Pipeline

In-depth data analysis:

• Data quality control;
• Peak calling and annotation;
• Fragment size distribution;
• Gene ontologies;
• KEGG pathways;
• Differential analysis.

Sample Requirements

Cell sample ≥ 50,000 cells; Nucleus sample ≥ 50,000 nuclei

Sample storage: Nuclei can be frozen at –20˚C for up to two days, avoid repeated freezing and thawing.

Shipping Method: Samples are supposed to stored in a 1.5 mL microcentrifuge tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: FastQ, BAM, coverage summary, QC report, full statistical analysis & alignments, custom analysis reports on customer request.

Demo Results

Case Studies

• • Identification of Transcription Factor Binding Sites (TFBSs) and Target Genes

    • The binding of TFs to specific DNA target sequences (or cis-regulatory elements) is the basis of the gene regulatory network. ChIP-seq is able to accurately determine the genome-wide distribution of TF binding sites in vivo. This is confirmed by studies of many plant transcription factors, which are involved in the regulation of development, biological clock, flowering time, hormonal signaling, abiotic or biotic stresses in different tissues or at different developmental stages in monocotyledonous and dicotyledonous plants.

      Depending on the protein, the number of recognized peaks varies from hundreds to tens of thousands. Depending on the genomic characterization, the positions of these peaks are annotated to sequences upstream of the transcription start site (TSS), 5 ' UTR, 3 ' UTR, exon or intron. Regions distributed near the TSS are promoter sites of genes and are considered targets of TFs. Regions distributed in the vicinity of the TSS are promoter sites of genes that are considered to be targets of TFs. In addition, the expression of these putative target genes can be further verified by RNA-seq.

      For more information, please refer to our article How to Identify Gene Promoters and Enhancers by ChIP-Seq.

• • Genome-Wide Histone Mark Detection

    • Histone marks (e.g. methylation and acetylation) play an important role in the regulation of gene expression during plant growth and development, as well as in response to different environmental stresses. ChIP-seq enables genome-wide detection of the distribution of modified histones in vivo.

• • Improvements in Sequencing Repetitive Genome Sequences

    • Heterochromatin or microsatellites have a large number of replicated sequences, which are difficult to sequence by conventional sequencing techniques. The presence of a large number of copies of mitotic sequences by tandem duplication, such as satellite DNA and reverse transcriptional transposons, can vary greatly in different species, even in organisms of close affinity, and their highly conserved functions are related to their epigenetic features, such as the histone H3 variant CENH3 in plants, which was identified by ChIP-seq using a CENH3-specific antibody to the ectopic genomic sequences present in new meristems of wheat chromosome 4DS.

      It can be seen that ChIP-seq can sequence repetitive sequence regions more efficiently, and the unique sequences flanking the repetitive sequences help align the reads to the genome.

FAQ

• • What are the controls when doing CHIP-Seq?

    • (1) Part of the DNA before immunoprecipitation (input DNA), this is commonly used;

      (2) DNA obtained by immunoprecipitation and not containing antibodies (mock IP DNA);

      (3) DNA obtained using non-specific immunoprecipitation methods.

• • Is ChIP-Seq applicable to all species?

    • The study should be performed on species that have a reference genome, genes spliced to the chromosome level, and complete annotation. Our technical team can also provide genome sequencing and de novo assembly services, please contact us for more details.

• • Does PCR after amplification during library preparation affect the final results?

    • ChIP-Seq based on the next-generation sequencing platform requires PCR during library preparation, mainly to obtain a sufficient amount of DNA for the on-board reaction, and the number of PCR cycles can be reduced if an adequate amount of DNA sample is provided. Therefore, the bias caused by PCR is very small.

• • Does the length of DNA fragments influence the outcome of subsequent sequencing?

    • Yes, quantitative accuracy, sequencing quality and data yield may be affected, and the larger the span, the greater the impact. Generally, we require that the main bands of interrupted DNA electrophoresis be in the range of 100-500 bp, with the main peaks in the range of 200-300 bp. Currently, the longest span allowed in the library construction process is 200 bp.

• • What is the standard analysis process of ChIP-Seq?

    • Firstly, the raw data is filtered, and then the clean data is aligned to the reference genome of the species, based on which the peaks are found. Based on the comparison results, peaks will be searched for, and then the peak-related genes will be functionally annotated, the differential peaks between samples will be analyzed, and the differential peak-related genes will be functionally enriched. In addition, the motifs of the peaks will be predicted.

• • How much data volume does ChIP-Seq typically require?

    • This depends on the size of the genome and how the factors of interest are combined (sharp regions for TFs and broad regions for histone marks). In mammals, at least 10-20 M is required for the identification of TFs and 10-45 M for broad histone marks, and the input control should be sequenced at the same depth as the ChIP sample. the number of reads also depends on the quality of the antibody and the efficiency of the immunoprecipitation. The higher the signal-to-noise ratio, the number of reads needed can be reduced appropriately.

References:

1. Massie CE, Mills IG. Mapping protein–DNA interactions using ChIP-sequencing. Transcriptional Regulation: Springer; 2012. p. 157-73.
2. Furey TS. ChIP–seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions. Nature Reviews Genetics. 2012;13(12):840-52.
3. Schmidt D, Wilson MD, Spyrou C, et al. ChIP-seq: using high-throughput sequencing to discover protein–DNA interactions. Methods. 2009;48(3):240-8.
4. Fullwood MJ, Ruan Y. ChIP‐based methods for the identification of long‐range chromatin interactions. Journal of cellular biochemistry. 2009;107(1):30-9.