GSM4405464: DNMT3AB_E6.5_Epi; Mus musculus; Bisulfite-Seq
Preparation of samples was performed as described in Smith, Chan, et. al, Nature 2014. The decidua of mated female mice were isolated on the morning of E6.5 and the conceptus removed. Then, under a stereomicroscope, the embryo was carefully bisected along the extraembryonic/embryonic axis, removing the ectoplacental cone from the extraembryonic ectoderm when apparent. After separation, Epiblast and extraembryonic ectoderm incubated for 15 min at 4ºC in 0. 5% Trypsin, 2.5% Pancreatin dissolved in PBS and filtered through a 0.2 mm syringe filter and allowed to rest for 5-10 minutes in KSOM at room temperature. Finally, visceral endoderm was removed by drawing the embryo through a narrow, flame drawn glass capillary and only embryo fragments with no apparent contamination were collected. On average, matched ExE and Epiblast from ~10 embryos were collected per assay. Samples were snap frozen in minimal volume prior to extraction and next-generation library synthesis. Genomic DNA for Whole Genome Bisulfite Sequencing was isolated according to Smith, Chan et. al, Nature, 2012 using a lysis buffer containing 300 ug/mL Proteinase K, followed by phenol:chloroform extraction and ethanol precipitation. Libraries were prepared using the Accel-NGS™ Bisulfite DNA library kit (Swift Biosciences) according to the manufacturers protocol. Final libraries were generated from 8-9 PCR cycles.