ChIP-seq (Chromatin Immunoprecipitation Sequencing), or DNA sequencing following chromatin immunoprecipitation, allows you to identify genomic regions associated with specific proteins, including transcription factors, and analyse the distribution of histone modifications. The method combines chromatin immunoprecipitation (ChIP) with next-generation sequencing (NGS).
The resulting data allow you to locate regions enriched in DNA associated with the protein under study, analyse potential mechanisms of gene regulation and compare protein-binding profiles and chromatin modification profiles between experimental conditions.
The service includes library preparation from the supplied ChIP DNA and sequencing of these libraries. We also offer sequencing of pre-made ChIP-seq libraries that you have prepared.
ChIP-seq workflow
This step is omitted if you have prepared the libraries yourself.
Applications of ChIP-seq
Technical specifications of ChIP-seq
- Platform: Illumina.
- Sequencing mode: as standard, PE150 (2 × 150 bp).
- Alternative configurations: single-end sequencing, including SE50, once availability and library compatibility have been confirmed.
- Input material: ChIP DNA or pre-made ChIP-seq libraries.
- Library preparation: from DNA following chromatin immunoprecipitation.
- Recommended amount of data: 10 Gb per sample in the standard PE150 configuration, with the option to adjust sequencing depth.
10 Gb corresponds to approximately 33 million PE150 read pairs with full-length reads. The actual number of fragments suitable for analysis depends on library quality, mapping efficiency and the read duplication rate.
Studies of broad regions of enrichment, such as those associated with selected histone modifications, may require a greater amount of data. We tailor the parameters to the characteristics of each project.
Sample Requirements
ChIP DNA for library preparation
| Parameter | Recommended requirement |
|---|---|
| Sample type | Purified ChIP DNA |
| Total amount | ≥50 ng preferred |
| Minimum amount | ≥10 ng, by agreement |
| Preferred concentration | ≥2 ng/µl |
| Volume | ≥25 µl |
| Fragment length | 100-750 bp |
| Preferred size range of the main fragment population | 200-600 bp |
| Purity (A260/A280) | 1,8-2,0 |
| Buffer | Nuclease-free water, EB or Low TE |
Sample acceptance is based on quality control. For smaller amounts of material, we need to confirm on a case-by-case basis whether library preparation is possible.
Pre-made ChIP-seq libraries
We offer sequencing of ChIP-seq libraries you have prepared yourself, without the need to repeat library preparation.
Libraries should be compatible with the Illumina platform and contain adapters and indexes that enable correct sequencing and sample identification.
We require the following information before accepting libraries:
- The kit used for library preparation.
- Concentration, volume and fragment size distribution.
- Index sequences and type (i7/i5).
- The planned sequencing configuration (SE or PE).
- The target number of reads or amount of data per sample.
Final requirements for library amount, concentration and volume are set according to the planned sequencing parameters. Libraries undergo quality control before being accepted for sequencing.
ChIP-seq bioinformatics analysis
The scope of ChIP-seq bioinformatics analysis may include:
- Sequencing data quality control and read filtering.
- Read mapping to the reference genome.
- Assessment of mapping quality, duplication and signal enrichment.
- Identification of enriched regions (peak calling).
- Annotation of regions relative to genes and regulatory elements.
- Analysis of signal distribution near transcription start sites (TSS).
- Identification of enriched sequence motifs (motif analysis).
- Functional analysis of genes associated with enriched regions, including GO and KEGG analyses.
- Comparison of enriched regions between experimental groups (differential peak analysis).
Depending on the project scope, results may include FASTQ, BAM, BED and bigWig files, as well as bioinformatics reports and visualisations.
We tailor the scope of analysis to the organism under investigation, the availability of a reference genome, the type of protein or histone modification, the number of biological replicates and the presence of appropriate control samples.
For projects involving only sequencing of pre-made libraries, bioinformatics analysis can be ordered as an additional part of the service.