GSM9824061: C103UI; Homo sapiens; RNA-Seq
HCO monolayers on chamber slides were washed with PBS containing 5 mM EDTA and dissociated with 200 µL/well Accutase (STEMCELL Technologies, 07920). Cells were mixed using wide-bore pipette tips and incubated at 37°C for ~55 min until fully digested. Wells from the same HCO donor line and treatment were pooled, neutralized with fetal bovine serum, and diluted with 1mL cold differentiation medium with ROCK inhibitor Y-27632. Then the cell suspensions were centrifuged at 300g for 5min at 4°C, resuspended in 100 µL differentiation medium with Y-27632, and passed through Flowmi Cell Strainers (Millipore Sigma, BAH136800040). The resulting single-cell suspension was adjusted to 1,000 cells/µL for sequencing. Single-cell gene expression libraries were generated using the Chromium Single Cell 3′ v3.1 kit (10x Genomics). Briefly, HCO single cell suspensions were combined with reverse transcription reagents, Gel Beads containing barcoded oligonucleotides, and oil on a Chromium X instrument (10x Genomics) to form Gel Bead-In-Emulsions (GEMs), enabling barcoded cDNA synthesis from each individual cell. Subsequently, the GEMs were disrupted and cDNA from each single cell was pooled. Following cleanup using Dynabeads MyOne Silane Beads, cDNA was amplified by PCR. The amplified cDNA was then fragmented, end-repaired, A-tailed, and ligated with sequencing adaptors. Final sequencing libraries were generated by amplification.