title: GSM9636660: day 27, 4D NMP, RA_BMP; Homo sapiens; RNA-Seq
library_construction_protocol: Single cell suspension from EBs is generated via papain dissociation method using the Worthington papain dissociation kit. Briefly, EBs are first collected in 15mL centrifuge tubes and washed twice with DPBS. The papain solution is prepared by mixing papain with DNAse, and the solution is allowed to activate for 10 minutes at 37ºC before adding to the EBs followed by triturating 10 times with a p1000 pipette. EBs are then preferably placed on an orbital shaker at 37ºC and 5% CO2 for 1 hour and 15 minutes and triturated 10 times every 5 minutes or until no visible EB clumps remain. Single-cell suspension of EBs are then centrifuged at 300 x g for 7 minutes. The cell pellet is resuspended in 2mLs of Ovomucoid protease inhibitor solution to stop the papain digestion and passed through a a 40µm cell strainer to remove undigested organoid debris. Single cells are then centrifuged once more at 300 x g for 7 minutes and the cell pellet is then resuspended in 1mL of 0.04% BSA in DPBS for GEM preparation using 10X genomics. ~ 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.