title: GSM8824005: Single cell RNAseq: Day 0; Homo sapiens; RNA-Seq
library_construction_protocol: SEAM cultures at Day 0, 14 and 28 were lifted in 0.05% Trypsin-EDTA (Life Technologies) followed by gentle mechanical dissociation in Accumax (STEMCELL Technologies). Dissociated cells were filtered through a 70 μm mesh membrane (Miltenyi Biotec) to achieve a single cell suspension. Magnetically labelled apoptotic and dead cells were removed using a Dead Cell Removal kit (Miltenyi Biotec) and MACS separation column. Cell concentration and viability were determined using a CellDrop FL Automated Cell Counter (DeNovix) prior to proceeding with either the 10X Genomics single cell protocol or total RNA extraction. Approximately 10,000 cells were used to generate a single cell library at each time point using the Chromium Next GEM Single Cell 3' Reagent kit (v3.1, Dual Index; 10X Genomics), following the manufacturer's protocol. Total RNA from two biological replicates from each time point were sent to Psomagen, Inc. (Rockville, MD) for library preparation and sequencing. Only samples with an RNA integrity number (RIN) ≥ 7 were considered for cDNA library preparation. cDNA libraries were generated using TruSeq Stranded Total RNA Gold Sample Prep Kits, (Illumina), which included an rRNA suppression step.