GSM9241771: 8 pooled human AP gastruloids at 120 hrs derived from hPSCs; Homo sapiens; RNA-Seq
For cell collection, human AP gastruloids (n=8) were harvested from the 96-well plate at 120 hours and pooled into a 15 ml centrifuge tube, washed with DPBS twice, and dissociated with 1ml 0.05% trypsin-EDTA (Thermo Fisher, 25300054) for 8 mins at 37 °C and quenched with 200 μL FBS. Gastruloids were dissociated into single cells by pipetting, pelleted by centrifuging at 300 rcf for 3 mins, resuspended in StemCell Banker (Amsbio, 11924), and stored at -80 °C. Single-cell RNA-seq (scRNA-seq) libraries were created using 10x Genomics Chromium platform, GEM-X Universal 3' Gene Expression v4 reagents kit following manufacturer specifications and guidelines. In brief, dissociated and frozen single cell suspension were thawed in Essential 6 medium containing 10% FBS, washed with PBS with 0.04% BSA, counted and diluted to 1500 cell/uL to target an optimal recovery of 20,000 cells/lane. The generated scRNA-seq libraries were visualized and quantified by TapeStation (Agilent), and sequenced on NextSeq 2000 (Illumina) with the following read structure: Read 1: 28 cycles; i7 Index: 10 cycles; i5 Index: 10 cycles; Read 2: 90 cycles.