GSM5550231: BS-seq_G9a_GLP_DKO_ExE; Mus musculus; Bisulfite-Seq
At E6.5, animals were euthanized and the uterine horn removed. Purified epiblast and ExE were isolated according to Ref. (Chenoweth and Tesar, 2010) with a few modifications. Briefly, E6.5 embryos were removed from deciduae and transferred to independent 25 μL drops of M2 media. Using a glass flame-pulled capillary, Reichart's membrane was reflected, and the embryo carefully bisected along the epiblast, ExE boundary. Then, each Epiblast/ExE pair was transferred into an individual drop of dissociation medium containing 0.5% trypsin and 2.5% pancreatin in PBS (w/v, Sigma). Embryos were cultured with slow orbital rotation at 4ºC for 15 minutes, after which they were transferred into new M2 drops. After ~5 minutes of resting, Epiblast and ExE were passed through a slightly narrower flame-pulled glass capillary to remove the visceral endoderm without disrupting the target tissue. Finally, each tissue was serially washed in 0.1% BSA in PBS prior to snap freezing in lysis buffer. Approximately 300-600 cells were collected from E6.5 embryos and directly transferred to 2.6 µl of Lysis Buffer (Takara Bio USA, Inc.) followed by snap-freezing at -80°C in preparation for cDNA synthesis using the SMART-Seq v4 assay. Full-length cDNA was prepared using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing and sequencing libraries prepared using the Nextera XT DNA library preparation kit (Illumina). The resulting libraries were evaluated using a 4200 TapeStation (Agilent Technologies) and quantified by qPCR. Libraries were pooled and sequenced on an Illumina NovaSeq SP or S1 flow cell using paired-end, 50 bp reads. The E6.5 ExE ∆G9a-Glp sample was isolated and processed into WGBS libraries using the Accel-NGS Methyl-seq kit as described in (Grosswendt et al., 2020) and sequenced on an Illumina NovaSeq using paired-end, 150 bp reads.