title: GSM6172319: B6.CAST, E6.5, ExE, H3K27ac, rep2 (ChIP-seq); Mus musculus; ChIP-Seq
library_construction_protocol: Natural timed matings were set up between C57BL6/Babr and CAST animals, and embryos were collected on embryonic day 6.5 (E6.5). ExE (N=8) samples were pooled (an estimated ~2500 cells), washed in PBS, and then flash frozen in 10µL of nuclear lysis buffer (Sigma).  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 (New England Biolabs) in prepared digestion buffer.  Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM 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.  Prior to the immunoprecipitation with antibody-bound beads, each chromatin sample was divided into 3-4 aliquots: one for each antibody and one 10% input control.  For each immunoprecipitation, 250ng of anti-H3K4me1 (Active Motif, 39298), 125ng of anti-H3K27ac (Abcam, ab4729), 250ng of anti-H3K4me3 (Diagenode K02921004), 125ng of anti-H3K27me3 (Millipore 07-449) or 250ng of anti-H3K36me3 (Diagenode C15410192)  antibodies were used.  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 solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio.  Library preparation was completed using the MicroPlex Library Preparation kit v2 (Diagenode) using Sanger 8-base indices for multiplexing, as per the manufacturers recommendations, and all libraries were amplified using 14 amplification cycles.  Libraries were purified with SPRI beads at a 1:1 ratio, and size and quality were assessed using a 2100 Bioanalyzer Instrument (Agilent) and Kapa library qPCR quantitation (Illumina).  Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer.  Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with 100mM 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.  Prior to the immunoprecipitation with antibody-bound beads, each chromatin sample was divided into 3 aliquots: one for each antibody and one 10% input control.  For each immunoprecipitation, 250ng of anti-H3K4me1 (Active Motif, 39298) or 125ng pf anti-H3K27ac (Abcam, ab4729) antibodies were used.  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 solid phase reversible immobilization (SPRI) purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio.  Library preparation was completed using the MicroPlex Library Preparation kit v2 (Diagenode) using Sanger 8-base indices for multiplexing, as per the manufacturers recommendations, and all libraries were amplified using 14 amplification cycles.  Libraries were purified with SPRI beads at a 1:1 ratio, and size and quality were assessed using a 2100 Bioanalyzer Instrument (Agilent) and Kapa library qPCR quantitation (Illumina).  Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500.