title: GSM9134260: cervicothoracic SCDH-INs_1; Homo sapiens; RNA-Seq
library_construction_protocol: Cell spheroids were manually removed from the MPS devices and singularized for scRNAseq by dissociation with Papain (Worthington). Using sterilized forceps, cell spheroids were removed from the PEG hydrogel mold, transferred to a conical tube containing PBS, and allowed to settle for 10 minutes. The spheroids were then resuspended in Papain solution containing DNase and incubated for 1.5 hours at 37° C on an orbital shaker. Cells were then triturated vigorously, centrifuged for 5 minutes at 300g, and quenched in ovomucoid solution containing DNase. Quenched cells were centrifuged, gently resuspended in PBS containing 0.2% BSA and 10 μM Y27632, then passed through a 40 mm cell strainer (Mitenyi Biotec) to remove debris. Cells were quantified and diluted to 700 cells/mL for sequencing.  Cells were kept on ice before being quantified and diluted for sequencing. Transcriptomic profiling was performed using the Chromium Single Cell Gene Expression system (10X Genomics), according to the manufacturer's recommendations using the Single Cell 30 Reagent v2/v3 kits (10X Genomics). Post-GEM-RT and post-cDNA amplification cleanup were performed using Dynabeads MyOne silane beads (Thermofisher) and SPRIselect (Beckman Coulter) kits respectively. Successful library preparation was confirmed using an Agilent Bioanalyzer (High Sensitivity DNA kit) and Qubit Fluorometer (High Sensitivity dsDNA kit).  Samples libraries were balanced for the number of estimated reads per cell and run on a NovaSeq 6000 system.