GSM8829202: Myf6-induced iMPC clone, MyoD-KO; Mus musculus; RNA-Seq
One MyoD-KO and one MyoD-WT iMPC clone at passage 4 were trypsinized and resuspended in PBS containing 2% FBS after inactivation with iMPC medium and centrifugation. The number of cells in each clone was manually counted with a hemocytometer. 12,000 cells were diluted with nuclease-free water and used for scRNA-Seq libraries preparation Libraries were prepared using the Chromium Next GEM Single Cell 3ʹ v3.1 (Dual Index) Kit (10x Genomics) according to manufacturer's instructions. Briefly, cells were loaded with barcoded Single Cell 3' v3.1 Gel Beads onto a Chromium Next GEM Chip G targeting the recovery of approximately 10,000 cells. After the Gel Beads-in-emulsion (GEMs) were generated, the gel beads were dissolved to release the uniquely-identifiable primers used for reverse transcription to produce barcoded cDNA from the polyA mRNA. GEMs were then broken to release the first-strand cDNA: this was then purified and further PCR-amplified. Amplified cDNA was then enzymatically fragmented, end-repaired, A-tailed, size selected, ligated to adaptors, indexed via PCR (Dual Index TT Set A, PN-1000215, 10x Genomics) and again size-selected. Diluted libraries were analyzed using the 4200 TapeStation System (Agilent Technologies, G2991BA) and D1000 ScreenTapes and reagents (D1000 ScreenTape, Agilent Technologies, 5067-5582; D1000 Reagents, Agilent Technologies, 5067-5583) according to manufacturer's instructions.