GSM6280646: EHT, Day 5 HTO cDNA; Homo sapiens; RNA-Seq
EHT cultures were initiated and carried out as described above. Three wells of a 96 well flat bottom TC-treated plate were prepared for each coating condition and cells were cultured in EHT media for 5 days. On day 5, non-adherent cells were harvested by gentle pipetting. Well replicates were pooled for each condition and pelleted by centrifugation at 300xg for 5 minutes. Samples were washed with 200 uL HF buffer (HBSS, Thermo Fisher Scientific, 14175103, supplemented with 2% Fetal bovine serum, Thermo Fisher Scientific, 12483020). Cells were stained with 0.1ug TotalSeq-B anti-human Hashtag antibody (Biolegend, 394633, 394635, 394637 or 394639) per sample diluted in 100uL HF buffer for 30 minutes on ice. Cells were washed 3 times with HF buffer. Cell density was determined by hand counting using a hemocytometer and samples were pooled at equal cell ratios per sample. The pooled sample was counted and loaded on the chromium controller as per the manufacturer's instructions aiming for a target cell capture of 5,900 cells. scRNAseq libraries were prepared using the 10x Chromium Single Cell 3' Capture v3.1 kit following the manufacturer's protocol