title: GSM3525814: CAST.B6_E6.5_Epi_Input_PBAT_3; Mus musculus; Bisulfite-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.   Prior to the immunoprecipitation, one 10% input  was taken from each chromatin sample for bisulphite sequencing. 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.  DNA samples were then bisulfite converted using the EZ DNA Methylation Direct kit (Zymo Research). The resulting DNA was purified using columns from EZ DNA Methylation Direct kit (Zymo Research). First strand synthesis was then performed using Klenow Exo- (New England Biolabs) with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This was followed by exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (ThermoFisher Scientific). Samples were then subjected to second strand synthesis similar to the first strand synthesis, followed by 12 PCR amplification cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA).  Samples were multiplexed using 75bp paired-end sequencing on Illumina NextSeq500.
attributes.tag: GEO Accession
attributes.value: GSM3525814