GSM6338673: hEE Day6; Homo sapiens; RNA-Seq
The extra-embryoids that exhibited an inner and outer compartment organization under brightfield are collected on day 4 and day 6 for single-cell isolation. Roughly 200 aggregates per time-points were serially washed with 0.1% BSA in PBS and briefly washed before incubation in 200 mL TrypLE (GIBCO) and incubation at 37ºC.  At five-minute intervals, structures were dissociated by pipetting under a stereomicroscope and returned to incubation until confirmed to be predominantly single-cell suspension.  Although the epithelial cells of the internal compartment dissociated readily, additional time was required to fully dissociate cells in the outside layer due to the appearance of a more extensive extracellular matrix.  Once dissociated, cells were collected in a total of 1 mL 0.1% BSA in PBS and filtered using a FlowMi™ cell strainer (40 mM, Scienceware®) and centrifuged for 5 min at 1,000 rpm.  After two washes, single-cell suspensions were resuspended at a set volume, counted using a standard hemocytometer, and adjusted to 1,000 cells per μL.  16,500 single cells for each time point were loaded onto a Chromium Controller (10x genomics) to generate single-cell transcriptomes using the Chromium Next GEM Single Cell 3ʹ platform according to the manufacturer's protocol (10x Genomics).