Assay summary for ChIP-seq_Mueller_lab_H3K4me3_0006AS
Mueller lab, University of Birmingham
Approximately 1500 embryos (dome/30% epiboly) or 200 embryos (prim-6) were dechorionated and fixed in 1.85% Formaldehyde in Hanks Media for 20 min at room temperature. Fixation was stopped using 1× Glycine followed by PBS washes (Wardle et al. 2006). ChIP experiments were carried out using the ChIP-IT Express Enzymatic kit (Active Motif) in line with manufacturer's instructions. In brief, embryos were resuspended in lysis buffer, incubated on ice for 20 min, and homogenized using a dounce homogenizer. Nuclei were resuspended in 200 μL digestion buffer. Chromatin was enzymatically sheared for 10 min at 37°C. The reaction was stopped, and 75 μL of sheared chromatin was used for ChIP reactions utilizing 4 μg of anti-H3k4Me3 (Abcam ab8580) or an equivalent volume of water as no antibody control. Samples were incubated overnight at 4°C while rotating. Magnetic beads were washed and decrosslinked for 4 h at 65°C. Samples were proteinase K and RNase A treated and purified using the QIAquick PCR Purification Kit (Qiagen) (dome/30% epiboly) or phenol chloroform extraction (24 hpf). Enrichment of target sequences was determined by qPCR using Power SYBR Green PCR Master Mix (Applied Biosystems). ChIP-seq was performed as described (Soler et al. 2011). In brief, 10 ng of ChIP DNA is end-repaired, ligated to single read adaptors, size selected, and amplified for 18 cycles according to Illumina's ChIP-seq protocol. Cluster generation is performed according to the Illumina Cluster Reagents preparation protocol (http://www.illumina.com). Samples were sequenced for 36 bp on the Illumina GA IIx platform or HiSeq 2000 system. The raw data from the Illumina Genome Analyzer are processed using the IPAR (Integrated Primary Analysis Reporting Software) and the Illumina Genome Analyzer Pipeline (GAP).