GSM8752680: Barcode Amplicon Sample 2.; Homo sapiens; OTHER
Cells were harvested by first washing with PBS (- calcium, - magnesium) followed by dissociating cultures into single cell suspensions with Accutase (StemCell Technologies, 07920) for 10 minutes at 37°C, cultures were then quenched with DMEM/F12 (Gibco, 11320033) and subsequently centrifuged at 300g for 3 minutes. The cell pellet was rinsed with PBS (- calcium, - magnesium) and centrifuged again at 300g for 3 minutes prior to library prep and resuspended in PBS-/- supplemented with 0.04% w/v BSA. scRNA-seq experiments were performed using 10x Genomics Chromium Single Cell 3' Reagent Kits v3 or v3.1 following the manufacturer's instruction. 5000 single cells were calculated as targeted input for each sample. For the first iteration, 10% of stem cells were spiked in as undifferentiated control. For the second iteration, 5% stem cells and 5% MG3.1-SFC were spiked in as undifferentiated and initial differentiation control. The only modification made to the protocol was at the Sample Index PCR step, where 5 µL of the PCR mix was taken out and mixed with 0.5 µL 1000X SYBR Gold (Invitrogen, S11494) for a qPCR reaction. The optimal amplification cycle was determined as the cycle just before half maximum of the total signal. Final libraries were sequenced on NextSeq 500 or NovaSeq with a goal of at least 30,000 reads per cell.