title: Single cell analysis of iPSC-derived cortical organoids in biofunctionalized gelatin hydrogels
design: Single cell analysis of iPSC-derived cortical organoids in biofunctionalized gelatin hydrogels
library_construction_protocol: The Worthington Papain dissociation kit was used to prepare organoids for single-cell RNA sequencing. Papain, DNAse, and ovomucoid inhibitor powders were re-suspended in Earle's media according to manufacturer specification. DNAse and papain solutions were gently mixed at a ratio of 1:10, and 1 μL of collagenase solution (100 units / μL in HBSS) was added. 5 - 8 organoids were pooled per well, and organoids were recovered from the hydrogels using collagenase. After washing the recovered organoids, the media wasexchanged for DNAse / papain / collagenase solution. The organoids were then incubated at standard tissue culture conditions (37°C, 5% CO2) for one hour. Afterwards, the organoids were dissociated by gently pipetting with a 1 mL pipette. The organoids were then returned to standard tissue culture conditions for an additional ten minutes. Using a 10 mL pipette, the digested tissue was then homogenized by pipetting up and down ten times. After letting the debris settle, the cell solution (without debris) was transferred to an ovomucoid inhibitor solution (5 mL Earle's media, 3 mL ovomucoid inhibitor) and spun down for 7 minutes at 300xg to pellet the cells. The supernatant was decanted, and the pellet was resuspended in cold 0.02% BSA in PBS. The cells were then pelleted and washed in 0.02% BSA on ice twice more before finally suspended to a final concentration before processing through the PIPseq method. Neural organoids were generated using the STEMdiff Cerebral Organoid Kit, with modifications. At day 0, iPSCs were aggregated in an ultra-low attachment 96-well plate (Corning / Nexcelcom), 9000 cells / well. Media was changed on days 2, 5, and 7. From days 2 - 7, cerebral organoid media was supplemented with dual-SMAD inhibitors SB43152 (Tocris 16-141-0) and LDN193189 dihydrochloride (Tocris 60-531-0). After embedding in custom biomaterials, organoids were kept in STEMdiff Cerebral Organoid Kit reagents (expansion media, days 7 - 10; maturation media, day 10 onwards) with no further modifications.  Cells were encapsulated using the PIPseq method. The PIPseq manufacturer's guide was then followed for nucleic acid extraction.For day 60 samples, the PIPseq T2 kit v4 chemistry was used. For day 90 and day 120 CC3 samples, the PIPseq T20 kit v4 chemistry was used. For day 90 KOLF samples, the PIPseq T2 kit v4PLUS chemistry was used. The PIPseq manufacturer's guide was then followed for library preparation.