ChIP-Seq

Sample Prep

• Test your antibody and sample prep before making a library.
• We recommend that the enrichment ratio (C+/I+)/(C-/I-) ≥ 10.
• C+ is ChIP at positive control
• I+ is input at positive control
• C- is ChIP at negative control
• I- is input at negative control
• To do this, you need two primer pairs, a positive control (+) and a negative control (-) which you measure on the ChIP and input samples using Q RT-PCR. See the figure to the right.
• We strongly recommend that you sequence an input library for each condition.  Note that in the figure, the input track has a strong peak at the same place as the ChIP peak.  The strength of inputs peaks or bias is dependent on the state of the cells as well as chromatin preparation conditions.
• Chromatin fragmentation is accomplished either via sonication or (for histones) DNase treatment.
• The sonication conditions can dramatically affect the results, so be consistent.

Sonication

• Sonication needs to be tuned to the sample.
• Successful sonication is a balancing act between a few opposing trends listed below.
• Some complexes are fragile so use a little sonication as possible.
• We only sequence the fragments with lengths of about 150bp, so enrichment in long fragments does not help.
• More sonication reaches deeper into dense chromatin.
• For low cell counts, you need to be as efficient as possible, so you need to leave as little material outside the fragment lengths that actually get sequenced.
• With the proper primers, you can verify enrichment in both the ChIPed chromatin and the library to ensure that the enrichment is still present in the library.

Sequencing

• ChIP-seq libraries are normally sequenced with 50bp SR runs on the hiSeq.
• ChIP-seq is primary a counting process, the reads only have to be long enough to place most of them uniquely in the genome
• 40 million reads are usually sufficient in a mammal.
• We have a multiplexed library protocol which can be used to put about 6 libraries in one lane.
• You may need more reads if the target protein is spread across large sections of the genome.

Initial Analysis

• ChIP-seq libraries are prone to PCR bias when the amount of starting material is small so we check the read redundancy of ChIP-Seq libraries.
• We align the reads to the genome with ELAND and keep the ones with a best alignment to just one position.
• We usually use HOMER to identify areas of enrichment, but can use other tools such as MACS or GLITR.
• We use the standard annotation pipeline to annotate the enriched regions.