How should I prepare and sequence samples for ChIP-seq?

If we prepare the sequencing libraries, we require the ChIP-seq DNA samples to be submitted de-crosslinked and sonicated.   The fragment length should best be between 100 and 300 bp. This will result in the tightest peaks. It is recommended to keep the fragment length under 500 bp in any case.
For ChIP-seq we sometimes need to start out with samples that are to low to measure the concentration.  Otherwise the general DNA sample recommendations do apply (buffer should be EB buffer or EBT; and more sample is certainly recommended if available.
General ChIP-seq recommendations would be:
  • The fragment length should be between 100 and 300 bp (up to 400 for the majority of molecules is acceptable).
  • Please make sure to run the input controls on the bioanalyzer or on an agarose gel beforehand and email us an image of these.
  • Sequence one “input control” per cell line/ sample type.
  • It is highly  recommended to verify the enrichment of your regions of interest (e.g. promoter regions) vs. the control samples by qPCR, before submitting the samples for sequencing.

The required read-number per sample will vary from target to target.  For the study of point source transcription factors the ENCODE project recommends to analyze at last 20 million (uniquely mapping) reads ( ).   Depending on the quality of your preps perhaps 75% of the reads can be expected to be uniquely mapping. ENCODE tends to err on the high side with their recommendations. Thus about 20 million reads per sample should be acceptable but this is likely the minimum number.

Category: 03 Sample Preparation, 04 Library Preparation and QC, 05 Sequencing

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