Assay summary for ChIP-seq_Cairns_Lab_H3K27me3_0002AS
Cairns Lab, HHMI
Sperm ChIP-Seq methods are as described previously (Hammoud et al. 2009 and Wu SF et al. 2011). For ChIP in early embryos, whole embryos were cross-linked with 2.2% formaldehyde for 15 minutes before quenching with 250mM glycine. Nuclei were then purified as described previously (Potok et al. 2013) and re-suspended in IP dilution buffer (16.7mM Tris-HCL, 167mM NaCl, 1.2mM EDTA, 1.1% Triton-X, 0.1%SDS). Lysates were then sonicated using a Branson Digital sonicator at 30% amplitude for 8 cycles of 10-pulse sonication (1 pulse per second) with 30sec rest on ice between each round of 10-pulse sonication. 5ug of antibody was then added to the lysate and left overnight to bind chromatin at 4C rotating. The following morning Protein A/G Dynabeads (ThermoFisher ID: 10003D) were bound to the lysate at 4C for 4 hours. Samples were then washed 4 times in RIPA buffer (50mM HEPES, 500mM LiCl, 1mM EDTA, 1% NP-40, 0.7% SDS) and eluted overnight at 65C in RIPA. DNA was then purified from eluate by phenol-extraction and alcohol precipitation. Libraries were made using the NEBNext ChIP-Seq Master Mix Set (New England Biolabs ID: E6240S). High- throughput sequencing was by IlluminaÕs protocol for 50bp single-end runs on an Illumina HiSeq 2500. See supplemental table for list of antibodies used.