title: GSM4083908: DKO.CAST_E6.5_ExE_Input_2; Mus musculus; ChIP-Seq
library_construction_protocol: Reciprocal natural timed matings were set up between C57BL6/Babr and CAST animals (denoted as B6/CAST and CAST/B6), and embryos were collected on embryonic day 6.5 (E6.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 +ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) and CAST males (denoted as DKO.CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated. E6.5 epiblast (N=4) and 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, as previously described (Brind'Amour et al. 2015 Nat Comm). 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 5 aliquots: one for each antibody, one 10% input and one 10% input for bisulphite sequencing. For each immunoprecipitation, 250ng of anti-H3K4me3 (Diagenode K02921004), 125ng of anti-H3K27me3 (Millipore 07-449) and 250ng of anti-H3K36me3 (C15410192) was 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 15 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.
attributes.tag: GEO Accession
attributes.value: GSM4083908