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Guidelines for ChIP-seq Projects

Chromatin immunoprecipitation (ChIP) sequencing enables an investigator to determine the distribution and abundance of DNA-bound protein targets across the genome. We have compared ChIP-seq library preparation kits from several different vendors and have concluded that the NEBNext ChIPseq Library Prep Reagent Kit delivers the strongest enrichment of peaks representing protein-bound chromatin. Following the completion of this comparison, the Core Facility adopted the NEBNext kit as the preferred protocol for ChIP-seq library preparation. Recommendations are provided below for preparing ChIP samples for library construction.
  1. Quantity of ChIP DNA for library construction: A minimum of 2-10 ng of ChIP DNA is required for constructing a sequencing library with the NEBNext ChIPseq Library Prep Reagent Set. If you have less than 10 ng, pool multiple ChIP samples together in a maximum volume of 20 ul.
  2. Purification of ChIP and Input Samples: Samples should be purified using a column based protocol such as the Zymo Research ChIP DNA Clean and Concentrator kit (cat# D5201) or the Qiagen QIAquick PCR purification kit (cat# 28104). Both of these kits enable elution of the ChIP DNA from the column in a volume as small as 10 ul. Avoid the use of organic compounds including phenol/chloroform for purification purposes as these reagents may adversely affect the enzymatic steps involved in library construction.
  3. Quantification of Sample: The concentration of ChIP samples can be measured by a Qubit dsDNA HS assay (Invitrogen cat#Q32851) or a pico-green assay. Measurement of ChIP samples on a NanoDrop tends to be inaccurate and misleading due to the low concentration of DNA and contaminants that may co-purify with the ChIP DNA sample. Quantification of the samples should be performed prior to verification of enrichment of known binding sites by qPCR.
  4. Number of sequence reads: The quantity of sequence reads required for a ChIP-seq experiment is dependent on the size of the genome and the number of binding sites for the protein of interest. For a higher eukaryote, 20-30 million reads is typically sufficient for analyzing transcription factor binding sites. In contrast, proteins with more binding sites such as RNA polymerase II or histones may require significantly more reads.
  5. Number of sequence reads: The quantity of sequence reads required for a ChIP-seq experiment is dependent on the size of the genome and the number of binding sites for the protein of interest. For a higher eukaryote, 20-30 million reads is typically sufficient for analyzing transcription factor binding sites. In contrast, proteins with more binding sites such as RNA polymerase II or histones may require significantly more reads.
  6. Microcentrifuge Tubes: We recommend the use of Eppendorf LoBind microcentrifuge (Fisher Scientific cat#13-6987-91) tubes for all steps of ChIP-seq experiments. LoBind tubes improve recovery by reducing sample to tube binding without the use of any coatings or additives. LoBind tubes are pcr-clean and likewise are RNase/DNase-free.
  7. Carrier DNA: Do not use salmon sperm DNA, calf thymus DNA or other DNA based carriers as a blocking agent at any step during the immunoprecipitation process. Carrier DNA that is added to your sample will function as template during Illumina library preparation and will contribute to the sequence reads along with your sample.
  8. Magnetic Beads: Dynabeads do not readily absorb random DNA from the immunoprecipitation cocktail and therefore are preferred over Sepharose, Sephadex etc.
  9. Formaldehyde Crosslinking: The concentration and incubation time of formaldehyde should be optimized by the user. As an initial guideline, a concentration of 1% formaldehyde for 5-15 minutes is common. Use Fisher Scientific catalog #BP531-500 or equivalent as stock source of formaldehyde.
  10. DNA Shearing: Investigators should aim for a majority of their crosslinked chromatin fragments to be in the 200-600 bp size range while ensuring the integrity of their protein. DNA fragments larger than 800 bp are sequenced less efficiently on a HiSeq instrument. Fragmentation has successfully been performed by researchers at the Huntsman Cancer Institute using either a Probe Sonicator or a Diagenode Bioruptor. Fragmentation should initially be performed as a time series to identify optimal time and power settings through analysis of the fragmented chromatin by agarose gel electrophoresis. The researcher should also define an optimal concentration of cells for shearing and follow that standard in each experiment. Variations in the quantity of cells can affect shearing efficiency.
  11. Epitope Monitoring: Fragmentation conditions can also be monitored by western blot analysis to access the enrichment of the protein that the ChIP Ab is directed against when compared to an input sample. Certain proteins are very susceptible to epitope destruction during shearing. Determine the time range that is optimal for fragmentation and also optimal for retention of protein.
  12. Positive Control: The H3K4Me3 antibody can be used as positive control for ChIP experiments and fragmented chromatin can be immunoprecipitated with this antibody alongside your Ab of interest.