Monday, 7 April 2014

Making ChIP-seq a little more robust

I had a fun time at Jason Carroll's group retreat in sunny Cromer a few weeks ago. I was invited to present some work we'd been doing on using Thruplex for ChIP-seq library prep and there was a lot of great science being discussed.

 
One of the presentations that stood out to me was from Kelly Holmes in Jay's group, she presented some simple steps all users can take to improve the quality of their ChIP-sequencing experiments: Quantify your Chromatin, Check your sonication, Standardise your library prep and Check your libraries. She also summarised a presentation on the use of controls and normalisation spike-in's.

I thought what Kelly had suggested was so simple and likely to have such an impact that I asked if I could share her talk with Core Genomics users, here are her 4 steps to ChIP-seq heaven:

1 Quantify your Chromatin
  • Quantify using nanodrop or similar.
  • Use 2.5ul of each sample after sonication.
  • Standardise the amount of Chromatin in your ChIPs.

2 Check your sonication
  • Run sonicated chromatin on e-gel or similar.
  • Reverse crosslink samples after sonication and before running on gel.
  • Check that majority of chromatin is below 1Kb.

3 Standardise your library prep
  • Use a single protocol across your samples.
  • If size-selecting keep fragment length the same.
  • Don't over-amplify your samples, determine the number of PCR cycles needed to get enough library for sequencing.

4 Check your libraries 
  • Once you've made a ChIP-seq library take some time over its QC an QT to get the very best results back from your sequencing lab.
  • QC and estimate library size using the Bioanalyser.
  • QT with qPCR using the estimated fragment size from above.
  • If libraries have different insert sizes then pooling can still be accurate, but fragment sizing is absolutely necessary

Once you've made libraries pool your samples so they are equimolar. We recommend normalising all samples to the same molarity (10-20) then pooling equal volumes to create a final pool at 10-20nM ready to submit and sequence.

Controls and normalisation spike-ins: Kelly also summarised a recent Actif Motif seminar at our Institute. Actif Motif sell ChIP-seq kits and antibodies as well as controls such as their Ready-to-ChIP HeLa Chromatin and qPCR control kits. At their recent seminar on ChIP-seq they presented the use of Drosophila chromatin as a spike-in control for histone mark normalisation: 750ng of drosophila chromatin in 30ug Human chromatin, co-IP for Drosphila specific protein and protein of interest, make ChIP-seq libraries and use Drosophila reads to normalise peaks for the protein of interest between samples.

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