GSM8447555: d25 GV H3K36me3_rep1; Mus musculus; ChIP-Seq
The ChIP-seq libraries for all samples were prepared using an ultra-low input native ChIP protocol, as previously described with the several modifications. Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease in prepared digestion buffer. Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with EDTA. Chromatin samples were precleared with Protein A/G beads rotating at 4°C for 2 hours and antibodies were bound to beads rotating at 4°C for 3 hours. Chromatin was added to the antibody-bound beads and rotated overnight at 4°C. This was proceeded by two low-salt washes, one high-salt wash, and the DNA was then eluted from the beads at 65°C for 1.5 hours. DNA from immunoprecipitated chromatin or input was purified using spri purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads at a 1.8:1 ratio. Library preparation was completed using the Diagenode MicroPlex Library Preparation kit v2, as per the manufacturers recommendations.