GSM9058150: DMSO; Homo sapiens; RNA-Seq
Culture medium was aspirated and 1 mL of warm 1 mg/mL Dispase (# 07923, Stem Cell Technologies) was added to each well of a 24-well plate containing a BME droplet with hFKOs. The droplets were then gently broken down by pipetting up and down using a treated 1,000-μl pipette tip (obtained by pipetting 1% BSA in PBS up and down five times in standard pipette tips) to prevent hFKOs fragments from sticking to the pipette tip. After a 40-min incubation at 37°C, the BME was dissolved and the hFKOs were transferred to 15-mL tubes containing ice cold DMEM F12, centrifuged, and resuspended in 3 mL 1x TrypLE express (#12605010, Gibco) supplemented with 10 uM ROCKi. The tubes were incubated at 37°C for a 10-min cycles, after which the hFKOs were disturbed by pipetting up and down 15 times and 10 μL of cell suspension was checked under a microscope to determine whether a single-cell suspension had been achieved. This process was then repeated until that was accomplished; two or three cycles are usually enough to break down the hFKOs to single cells without affecting cell viability. hFKO cells were (harvested, washed and) resuspended in PBS supplemented with 0.5% BSA to achieve an optimal concentration of approximately 1000 cells/μL for loading onto the Next GEM Chip (10x Genomics). Libraries were prepared using the 10x Genomics Chromium Controller in conjunction with the single-cell 3' v3.1 kit, protocol revision E. cDNA synthesis, barcoding, and library preparation were carried out according to the manufacturer's instructions. Briefly, cDNA amplification was performed for 11 cycles and sample index PCR was performed for 12 cycles using Chromium i7 Sample Indices. The resulting libraries were quantified and analysed with Qubit and TapeStation and were sequenced on the NextSeq 500 platform (Illumina), following the manufacturer's protocol, using a NextSeq 500/550 High Output Kit v2.5 (75 cycles) kit (Illumina).