title: GSM9636541: 4D NMP, day 0; Homo sapiens; RNA-Seq
library_construction_protocol: Worthington's Papain dissociation system was used to generate single cells in 0.04% BSA in PBS from 3D cultured embryoid bodies (EBs). EBs are first incubated at 37˚C with 20 units/mL papain and 0.005% DNase solution and triturated every 10 minutes for about 1 hour until tissue is fully dissociated. To stop the dissociation the dissociated cells are spun down and resuspended in EBSS, albumin-ovomucoid inhibitor, and DNase. Cells are then passed through a 40μm filter and spun down to be resuspended in 0.04% BSA in PBS. ~ 10,000 live cells/condition were used to generate single-cell cDNA libraries using protocol described by the 10X Genomics 3'GEX library preparation kit. Cells were first partitioned into GEMs (gel-beads-in-emulsion) containing using the 10x Genomics Chromium controller. Further, 3' mRNA-seq gene libraries were prepared using the Chromium Single Cell 3′ Library & Gel Bead Kit v2 (10x Genomics), according to the manufacturer's instructions. Library quality control was assessed using Agilent and Qubit assays. If quality control is within acceptable conditions (e.g., library concentration of 20-40ng/ul), libraries were sequenced on the Illumina NovaSeq X Plus 10B. For 3'GEX libraries targeting 10,000 cells 1,800 million reads/sample were obtained.