GSM6123268: H1-D39_S8; Homo sapiens; RNA-Seq
At D11, D14, D21, D32, D39 of differentiation, cells were collected in microcentrifuge tubes and washed with PBS. Accutase® was used to dissociate aggregates into single cells, which were then stained with propidium iodide (Sigma) in a PBS solution containing 0.2% BSA.  Approximately 200,000 live cells (propidium iodide-negative) were sorted with a FACSAriaTM Fusion Flow Sorter at a sorting speed lower than 3,000 events per second to minimize damage to the cells. Sorted cells were pelleted with 250 x g for 5 minutes at 4°C, and counted with a Scepter™ automated cell counter. 10,000 accurately counted cells per sample were loaded onto a 10X Chromium Controller for GEM formation and cell barcoding using Next GEM Single Cell 3' v3.1 reagents. Barcoded single cells were subjected to cDNA synthesis and sequencing library construction using 10X Next GEM Single Cell 3' v3.1 reagents according to manufacturer's instructions. Final libraries were quantified using a Qubit fluorimeter (Life Technologies) and the fragmented cDNA was verified using a Tapestation (High Sensitivity D1000, Agilent). Libraries were sequenced on NextSeq 500, HiSeq 4000 or NovaSeq 6000 sequencers (Illumina) and reads were trimmed afterwards to fit into corresponding analysis pipeline. Single cell RNA-seq with 10X Next GEM Single Cell 3' v3.1 kit