GSM1932419: E16_MS08T77; Macaca fascicularis; RNA-Seq
For single cell isolation from cy pre-implantation embryos, a whole embryo was incubated with 0.25% trypsin/PBS  for around 10 min at 37℃, then dissociated into single cells by repeated pipetting, and dispersed in 0.1 mg/ml of PVA/PBS  for preparation of single-cell cDNAs. For single cell isolation from cy post-implantation embryos, the implantation site was dissected out from the uterus and the embryonic fragment containing the epiblast (EPI), amnion, hypoblast, and yolk sac endoderm was isolated manually. The fragment was incubated with 0.25% trypsin/PBS for around 10 min at 37℃, then dissociated into single cells by repeated pipetting, and dispersed in 0.1 mg/ml of PVA/PBS. For single cell isolation from mouse E5.5 and E6.5 embryos, the embryos were dissected out from decidua and the extra-embryonic ectoderm was removed manually. The EPI/visceral endoderm (VE) were incubated in 0.25% Pancreatin/0.5 % Trypsin/Polyvinylpyrrolidone, and EPI and VE were separated by mild pipetting. For E5.5 embryos, EPI and VE were incubated with 0.25% trypsin/PBS separately for around 10 min at 37℃. For E6.5 EPI, their proximal parts were dissected out manually, and the proximal EPI and whole VE were incubated with 0.25% trypsin/PBS separately for around 10 min at 37℃. The proximal EPI and VE were dissociated into single cells by repeated pipetting, and dispersed in 0.1 mg/ml of PVA/PBS. For single cell isolation from cyESCs, cells were first detached as clumps with CTK solution, incubated in 0.25% trypsin/PBS (Sigma-Aldrich) for around 10 min at 37℃, and dispersed into single cells in 1% (vol/vol) KSR/PBS containing 10 M of the ROCK inhibitor Y-27632. Cells under feeder-free condition were directly incubated in TrypLE Select for around 5 min at 37℃, and dispersed into single cells in 1% (vol/vol) KSR/PBS containing 10 M of the ROCK inhibitor Y-27632. For single cell isolation from mESCs and EpiLCs, cells were incubated in TrypLE Select for around 5 min at 37℃, and dispersed into single cells in 1% (vol/vol) KSR/PBS. For SC3-seq analysis, cDNA synthesis / amplification from single cells and library construction from the cDNAs were performed as described previously [Nakamura et al. 2015, NAR, 43, e60].
GEO Accession
GSM1932419