GSM8079007: RPM_2D_WT; Homo sapiens; RNA-Seq
Single-cell capture, reverse transcription, cell lysis, and library preparation were carried out by the WCM Epigenomics Core Facility using Chromium Single Cell 3' v3 kits according to the manufacturer's protocol (10x Genomics, USA). Single cell suspenions of RUES2-dervied RPM and RPM (T58A) day 50 cultures were prepared by incubating wells in 0.05% Trypsin/0.02% EDTA for 10-15 min. Digestions were then quenched with excess 0.1% BSA/PBS buffer and passed through 40μm mesh strainer. EGFP+ singlets were sorted at the WCM Flow Cytometry Core Facility on a BD Influx or Sony MA900 cell sorter. Each RUES2-dervied RPM and RPM(T58A) day 50 culture pools were isolated from 3 wells of a 12-well plate across two biological replicates, targeting ∼50,000 viable, EGFP+ viable cells per replicate. Cell counts were then adjusted to 6000-9000 cells/50uL in PBS by Trypan Blue exclusion to achieve an estimated capture of 4000-5000 cells. Sequencing was performed by the WCM Genomics Resources Core Facility on a HiSeq 2500 (Illumina, paired-end protocol with 26 base pairs for read 1 and 98 bases for read 2). Single cell RNA sequencing from RP cultures were obtained from previous experiments (5). Transcript abundance was quantified by mapping sequencing reads to a custom human transcriptome reference (GENCODE v42, hg38) that included EGFP and was performed using simpleaf/alevin-fry (12). 10x v3