GSM8634926: 1_CTRL, replicate 1, scRNAseq; Mus musculus; RNA-Seq
Respective number of gastruloids of the four different conditions at 96hpa were collected from 96-well plates and transferred to a 15ml falcon (control: n=50; 2-DG, 2-DG+ActA, 2-DG+CHIR: n=75). Medium was removed and gastruloids were washed with prewarmed PBS -/- (Sigma, D8537). PBS-/- was removed and 1ml TrypLE (Thermo Fisher Scientific, 12604) was added. Samples were incubated for 5min at 37°C with repeated flicking. Cell suspension was then gently pipetted up and down to ensure a single-cell suspension. Subsequently, 4ml of ice-cold 0.1% RNAse-free BSA in PBS-/- (BPBS) was added and cell suspension was centrifuged at 170g for 3min at 4°C. All following steps were performed on ice/at 4°C. Cells were washed 2 times in BPBS, before they were resuspended in 300µl BPBS using a wide bore P1000 tip. Cell suspension was then passed through a cell strainer (Corning, 352235), counted and diluted to a target concentration of 1.0-1.5x106 cells/ml. Library preparation and sequencing was performed by the CRG Genomics Unit. Cells were barcoded using a Chromium GEM-X Single Cell 3' Reagent Kits v4 (10x Genomics) according to manufacturer's instructions. Libraries were sequenced with a NovaSeq 6000 system (Illumina).