GSM9128519: Organoid A, 75% hgECM; Homo sapiens; RNA-Seq
Organoids were disassociated into single cells using trypsinization or cold enzyme protease digestion. Trypsinization: organoid pellets were washed in 1 mL of ice-cold PBS per well, then centrifuged at 200 g for five minutes. HGO pellets were then treated with 350 µL of 0.25% trypsin EDTA for ten minutes at 37°C. Next the trypsin was diluted with 600 µL of cold DMEM, then the pipetted 100 times, to further disassociate the HGOs. The organoid cells were then washed with 5 mL of ice cold DMEM, then centrifuged for 7 minutes at 300 g. The organoid cells were then washed two additional times with ice cold DMEM, to remove any remaining Matrigel or hgECM. Ccold protease digestion: HGOs were washed with 1 mL of ice-cold PBS per well, then incubated for one hour on a shaker at 4°C in 2 mL of ice-cold PBS with 4 mg/mL native Bacillus licheniformis protease, 10 µg/mL DNase, and 5 mM CaCl2. The cell mixture was vortexed for 30 seconds every 20 minutes. After digestion, the cell mixture was filtered through a 60 µm filter, then washed twice with 5 mL of ice-cold DMEM and centrifuged at 300 g for 10 minutes. After disassociation, the total cell concentration and viability were determined using a hemocytometer and trypan blue staining. Organoids A and B: Library preparation followed 10X Genomics protocols for Next GEM 3' kit V3.1 with no modifications. Organoids A and B: Library preparation followed 10X Genomics protocols for GEM-X 3' kit V4 with no modifications. Briefly, dissociated organoids were encapsulated by partitioning oil with reverse transcription master mix and gel beads containing barcode sequences using either the Chromium Controller (organoids A and B) or Chromium X (organoids C and D) machine from 10X Genomics. Poly-A RNA was ligated to the 10X barcodes and reverse transcribed. Barcoded cDNA was then amplified, fragmented, size-selected, and prepared for Illumina paired-end sequencing.