YOU ARE HERE PUBLICSCIENTIST WEBSITE

Whole genome amplification protocol for ChIP-chip (Prot 30)

Henriette O'Geen and Peggy Farnham

Introduction

The technique of chromatin immunoprecipitation (ChIP) has proven to be a powerful tool, allowing the detection of protein-DNA interactions in living cells. Hybridization of ChIP samples to DNA microarrays (i.e. the ChIP-chip assay) allows a global analysis of binding sites for transcription factors and components of the transcriptional machinery, as well as of chromatin modification patterns. However, a single ChIP sample does not yield enough DNA for hybridization to a genomic tiling array. Therefore, we have adapted the standard protocol for whole genome amplification using the Sigma GenomePlex WGA kit to amplify our ChIP sample (O'Geen et al., 2006). Using Oct4 ChIP-chip assays as an example, we have compared the quality of ChIP-chip data derived from 1) WGA amplified ChIP samples, 2) a pool of 10 ChIP samples without further amplification, and 3) linker-mediated PCR (LMPCR) amplification of ChIP samples. Based on the low background, reproducibility, and the fact that a single WGA amplified ChIP sample can provide sufficient material for several array hybridizations, we recommend the WGA protocol for ChIP-chip analyses. We have successfully tested our new ChIP amplification protocol on a variety of different factors (E2F family members, KAP1, CtBP2, ZNF217) as well as on histone modifications (H3me3K9, H3me3K27, H3me3K4) (Krig et al., 2007; O'Geen et al., 2007). Another benefit of the WGA amplification method is the ability to perform a second round of amplification from the initial WGA product if a higher DNA yield is required. We have applied the re-amplification protocol to KAP1 amplicons that were hybridized to a whole genome tiling array set consisting of 38 arrays (O'Geen et al., 2007). Detailed protocols for ChIP assays from mammalian cells and tissue samples, as well as preparation of amplicons can be found on the Farnham Lab website.

PDF version

Henriette O'Geen and Peggy Farnham

University of California Davis - UC Davis Genome Center - Genome and Biomedical Sciences Facility - 451 East Health Sciences Drive - Davis, CA 95616-8816, USA

Henriette O'Geen and Peggy Farnham
Submit to FacebookSubmit to Google PlusSubmit to Twitter