GSM8666087: elongating heart organoid, day 5; Homo sapiens; RNA-Seq
eHOs were pooled into a 15 mL conical tube. Organoids were washed with PBS and incubated in pre-warmed 10x TrypLE Select Enzyme solution (Gibco) for 20 minutes at 37°C, with gentle pipetting every minute. To reduce sheer stress on the cells, pipette tips were trimmed. Dissociated cells were transferred to 5 mL of pre-warmed N2B27 medium, centrifuged 200 x g for 5 minutes, and resuspended gently in cold PBS for further processing. Libraries were prepared using the Chromium controller according to the 10x chromium Next GEM Single Cell 3' v3.1 protocol (CG000315). Briefly, the cell suspensions were diluted in nuclease free water to achieve a targeted cell count of 10,000. Cell suspension was mixed with master mix and loaded with Single Cell 3'V3.1 Gel Beads and Partitioning Oil into a chromium Next GEM chip G. RNA transcripts from single cells were uniquely barcoded and reverse transcribed within droplets. cDNA molecules were pooled and the cDNA pool then go through an end repair process, the addition of a single 'A' base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library. The purified libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide(KAPA) and qualified using the Agilent Technologies 4200 TapeStation.