Introduction

ChIP-exonuclease (ChIP-exo) is a high resolution method of genome-wide mapping of DNA-associated proteins that outperforms ChIP-seq by all parameters. We adapted this method created by Frank Pugh and Ho Sung Rhee from SOLiD to the Illumina sequencing platform (Rhee and Pugh, 2011; Sérandour et al, 2014) (cf figure 1). We provide here a ChIP-exo protocol, optimized on 60 million cells (4 dishes of 15 cm diameter at confluence). Lower cell number has not been tested.

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Introduction

The discovery of 5-methylcytosine oxidative products led to the development of a large number of techniques to allow the detection of cytosine modifications and to map them genome-wide. Here is described an antibody-based method to detect 5-hydroxymethylcytosine (5-hmC) in mammalian cells. This protocol consists of an extraction of genomic DNA followed by a three-day immunoprecipitation procedure. 5-hmC containing immunoprecipitated DNA fragments can be further analysed by qPCR or submitted to high throughput sequencing. It is important to note that this approach allows the mapping of 5-hmC only at low resolution and, as a consequence, other techniques must be used for single nucleotide resolution.

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Introduction

Zebrafish (Danio rerio) is well established as a model organism to study embryogenesis. Practical advantages of using zebrafish are that hundreds of synchronized embryos can easily be collected, embryos are transparent, development is rapid and external, and its genome is sequenced. The 9^th assembly of the zebrafish genome (Zv9) reports 1.41 billion base pairs with ~24,000 protein-coding genes. Information on the Danio rerio genome assembly can be found at http://www.sanger.ac.uk/Projects/D_rerio/Zv9_assembly_information.shtml.

A unique feature of zebrafish (and of anamniote vertebrates) is a developmental period of several hours after fertilization in the quasi-absence of on-going transcription. In zebrafish, this developmental period lasts for 3.3 h during which the embryo undergoes 10 rounds of synchronous Chromatin Immunoprecipitation Assay for Early Zebrafish Embryos (Prot59) cell divisions. Zygotic genome activation (ZGA) occurs at the ~1,000-cell stage, at the mid-blastula transition (MBT) (Tadros and Lipshitz, 2009) (see http://www.neuro.uoregon.edu/k12/Table%201.html for a description of zebrafish developmental stages). This 3.3 h pre-MBT period provides a unique opportunity to identify epigenetic processes, including enrichment in post-translationally modified histones, associated with the establishment of the embryonic gene expression program (Lindeman et al., 2011). [...]

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Introduction

RNA polymerase II (RNAPII) is responsible for the transcription of protein-coding genes, in addition to a large number of non-coding RNAs. The C-terminal domain (CTD) of its largest subunit consists of multiple heptad repeats (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7; 52 copies in mammals) that are targeted for a wide range of post-translational modifications, providing a platform for interaction with chromatin modifiers and RNA processing machinery (Brookes and Pombo, 2009). Ser5 residues become phosphorylated during transcription initiation and Ser2 residues during productive transcription elongation. [...]

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¹ Genome Function Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.
² Present address: Cancer Research Technology Discovery Laboratory, Wolfson Institute for Biomedical Research, The Cruciform Building, Gower Street, London, WC1E 6BT, UK.

Corresponding author: Ana Pombo, MRC Clinical Sciences Centre, Imperial College School of Medicine,Hammersmith Hospital Campus, Du Cane Road, London W12 0NN,UK.

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.

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