GSM6894029: D5, pool A, replicate 1; Homo sapiens; RNA-Seq
For each time-point, cells were detached using TrypLE-express (ThermoFisher, Gibco 12604). Harvested cells were then centrifuged at 300 g and resuspended at the final cell density of 100 cells/mL using a solution containing 40% KnockOut Serum Replacement (KSR, ThermoFisher, Gibco 10828) in DMEM. For each timepoint, two replicates were produced, each containing cells from 4 independent chips that were pooled together then divided in aliquots containing 5,000-80,000 cells. Samples were cryopreserved in DMEM supplemented with 40 % KSR and 15 % DMSO and stored in liquid nitrogen. scRNA-seq libraries were generated using one or two samples for each replicate. Briefly, each cryopreserved aliquot was thawed at 37 °C until a tiny ice crystal remained in solution. Then each sample was diluted under gentle shaking by dropwise adding 10 volumes of DMEM supplemented with 40 % KSR. Cells were washed twice using a washing buffer containing 8 % MACS Running Buffer (Miltenyi, 130-091-221) in PBS. Cells were then resuspended in the washing buffer and filtered through a 40 µm cell strainer (Biosigma, 010198Z). Cell viability and concentration were checked by visual inspection using Trypan Blue (Logos Biosystems, L12002). 10X scRNA-seq 3' gene expression