GSM5220797: 21_I_epi_Eed; Mus musculus; RNA-Seq
At E6.5, animals were euthanized and the uterine horn removed. Purified epiblast and ExE were isolated according to Chenoweth, J. G. et al. 2010 Methods Mol. Biol. with the 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% tryspin 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 from epiblast and ExE 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 flow cell using paired-end, 50 bp reads.