ChIP sequencing (ChIP-Seq) has revolutionized the way investigators study DNA-protein interactions by enabling the genome-wide analysis of protein binding. Genomic regions that interact with the protein of interest are detected and quantified by sequencing the subset of the genome that is co-immunoprecipitated with that protein. The Genome Sciences Resource uses state-of-the-art technology and methodology to produce high quality ChIP-seq data sets. Starting from investigator-supplied immunoprecipitated DNA, indexed sequencing libraries are constructed using the Illumina TruSeq ChIP sample prep kit and sequencing is conducted on the Illumina HiSeq 2500.
The immunoprecipitation step in a ChIP-Seq experiment produces the enriched population of DNA for identification by sequencing, and VANTAGE strongly recommends that the investigator validate this enrichment prior to sequencing. This is accomplished using quantitative PCR of a known binding region to compare it's relative abundance in positive (target antibody) and negative (control IgG) precipitations.
Project planning and consultation
We recommend that investigators consult with VANTAGE staff prior to submitting samples for ChIP-Seq. The total cost and the interpretability of a ChIP-Seq experiment depend on parameters that vary greatly between projects, and this consultation allows VANTAGE staff and the investigator to more accurately assess them. The most important factors are the genome size of the experimental system, the expected size of the targeted regions, and the quality of the immunoprecipitation.
Each sequencing library requires a minimum of 5 ng of immunoprecipitated DNA fragmented to a 150-300 bp size range. VANTAGE will verify the concenration and size range of each sample.
It is important to use properly size fragmented DNA as the starting material: larger DNA fragments will result in greatly reduced yields and poor sequencing performance. The size range of DNA fragments in each new chromatin lysate should be verified by the investigator. This can be accomplished using agarose gel electrophoresis. VANTAGE will verify the size range of each submitted sample using the Agilent bioanalyzer High Sensitivity DNA chip
Delivery of ChIP-Seq results
After data collection and demultiplexing are completed and the results have been checked by VANTAGE staff, the investigator is notified by email. This notification includes directions to download the FASTQ files from VANTAGE LIMS. Customers may also elect to obtain FASTQ files from VANTAGE on an external hard drive.
Library and Data Retention
Sequencing libraries produced by VANTAGE will be maintained as a frozen stock for a minimum of 3 years. Investigators may purchase additional sequencing from any of their VANTAGE libraries. Primary sequencing data (gzip-compressed FASTQ or BAM format) is maintained on the Vanderbilt Bluearc data depot for 90 days after data release.
Sequencing Quality Control
The quality and integrity of sequencing results are monitored for each data collection run and for each sample. A positive control is incorporated into every sequencing run and it's performance is tracked by VANTAGE to ensure performance. The sequence yield is evaluated for each sample and supplementary data is collected for any sample that does not meet the customer-specified yield.