GSM4007644: Day 14 Dorsal patterned Cells round 3; Homo sapiens; RNA-Seq
MiSTR tissue was divided into two halves along the gradient, one side used to collect the five A-E samples for mRNA analysis. The other side was dissociated into a single-cell suspension using Accutase (Thermofisher), for day 0, day 1 and day 2 samples, or with Neural dissociation kit (P) on a GentleMACS dissociator (both from Miltenyi Biotec) for day 14 samples. The cells were then frozen in CS10 CryoStor (Stemcell Technologies) and stored at -150oC until needed. In the meantime, each sample underwent mRNA analysis for quality control selection for scRNA-seq. Cryopreserved cells, quality control cleared, were thawed, counted and mixed in equal numbers into a final single-cell suspension of 350 to 500 cells/ml solution in 0.01% BSA in PBS. For 0 to 48h tissues, cells from 3 biological MiSTR tissue replicates were pooled prior to sequencing. For day 14 tissues, the sequenced samples contained a mix of cells from regions A to E (n=1-3 biological replicates per region). A total of 9000-17000 cells were loaded per lane onto the 10X Chromium (10X Genomics) . Chromium Single Cell 3' Reagent Kits (v2 Chemistry)