GSM4116261: Mouse ESC Day 2; Mus musculus; RNA-Seq
For generating cell suspensions from mouse embryo tailbuds, E9.5 embryos (25-28 somite stage) from CD-1 IGS mice (Charles River) were collected and the posterior part of the embryo, including the last 3 pairs of somites, was carefully dissected from two littermate embryos and subsequently processed as separate samples. Tissues were collected in PBS and dissociated in TrypLE Express for 10 min at 37ºC. Cells were rinsed in PBS/EDTA, transferred to 0.5% BSA in PBS, mechanically separated by trituration and run through a 30 µm cell strainer. Cells were spun down at 200 g for 4 min at 4ºC and resuspended in 100 µl 0.5% BSA in PBS. For generating cell suspensions from human IPSC differentiated cultures, 3x10^4 MSGN1-Venus cells were seeded on Matrigel-coated 24-well plates 48 hours prior to differentiation. Cells were differentiated as described. All samples (days 1-4 and hiPS control samples) were dissociated, collected and captured on an inDrops setup on the same day, two biological replicates per sample. For dissociation, cells were briefly rinsed in PBS, and incubated in TrypLE Express (Gibco) for 5 min at 37ºC. Dissociated cells were run through a 30 µm cell strainer, spun down at 200g for 4 min at 4ºC and resuspended in 100 µl 0.5% BSA in PBS. For generating cell suspensions from mouse ESC differentiated cultures, 1x10^4 pMsgn1-Venus cells were seeded on fibronectin-coated 6-well plates and differentiated as described. Samples for days 0 and days 2-5 were dissociated in TryplE Express (Gibco) for 3-10 minutes, washed several times in PBS, passed through a 40 µm cell strainer and resuspended in 0.1% BSA in PBS with Opti-Prep at a final density of 200,000 cells/ml. All samples were dissociated, collected and captured on the same day in biological duplicates. Single-cell RNA-Seq libraries were prepared using a protocol customized for the inDrops platform (see Zilionis et al., Nature Protocols 2017) Single-cell RNA-Seq (inDrops V3)
GEO Accession
GSM4116261