GSM4084100: B6.CAST_E7.5_Epi_PBAT_1; Mus musculus; Bisulfite-Seq
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 7.5 (E7.5). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each embryo were manually separated.  Individual E7.5 epiblast and ExE samples were washed in PBS, and then flash frozen in 10µL of RLT+ buffer (Qiagen). Samples were digested at 37°C for 1 hour.   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). The bisulfite conversion reaction was repeated due to poor initial conversion.  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 10 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 150bp paired-end sequencing on Illumina NextSeq500.