GSM7035398: scRNA-seq of the whole wt stembryos at 96h, rep1; Mus musculus; RNA-Seq
48 to 96 gastruloids were collected 96h or 120h after aggregation and washed twice in 1ml of PBS. Gastruloids were dissociated in 100ul of Stempro Accutase© for 5 minutes at 37°C, full dissociation was ensured by mechanical dissociation (pipetting) and verified to ensure absence of doublet cells. Then gastruloid cells were washed twice in 500ul PBS, and resuspended in an adequate volume of PBS 0.04%BSA at 106 cells/ml. All centrifugation steps were done for 5 minutes at 350G in DNA low binding Eppendorf tubes. Single cell suspensions were filtered using Flowmi Cell Strainer 40um and then were subjected to single cell RNAseq using the 10x genomics platform (V3, and V3.1 chemistry) following manufacturer's recommendation. cDNA preparations were performed according to 10x Genomics recommendations and was amplified for 10 to 12 cycles. And sequenced on a Hiseq 4000 with the cbot2 chemistry. At least 2 replicates were performed for each time points. In addition, a third 120h timepoint was generated by dissection in two portions to separate the anterior and posterior halves and each portion was analyzed separately by single cell RNAseq.