GSM7916615: LBC_T2, scRNAseq; Homo sapiens; RNA-Seq
For the sample coresponding to the passage day, adherent and floating cells were sequenced separately. Each sample was pelleted by centrifugation at 300x g for 5 minutes. Replicate wells were pooled together and washed with PBS. Samples were Fc blocked (BD, 564220) and labelled with zombie-UV viability dye (BioLegend, 423108) on ice in the dark for 20 minutes. Next, each sample was labelled with 0.1 ug of a cell hashing antibody (TotalSeq-B anti-human Hashtag antibodies 1 through 8, BioLegend). Cells were labelled for 30 minutes on ice in the dark. Next, samples were washed 3 times with HF buffer and counted prior to pooling. Samples were combined in to two pools, one for the EHT time points and another for the progenitor T cell differentiation time points. To enrich for Zombie-UV-negative viable cells, the EHT time point pool was subject to Fluorescent Activated Cell Sorting (FACS) using a MoFlo Astrios (Beckman Coulter). Cell pools were counted and loaded on the Chromium Controller (10x genomics) using the Chromium Single Cell 3' Reagents kit (v3.1) and libraries were generated according to the manufacturer's instructions. Libraries were sequenced on the NextSeq2000 (Read 1: 28bp Index1: 10bp Index 2: 10bp Read 2: 90bp)