GSM9455481: WT; Homo sapiens; RNA-Seq
Briefly, 3-month-old organoids were dissociated using 4 ml StemPro Accutase™ Cell Dissociation Reagent (Thermo Fisher Scientific, A1110501) for 12 minutes at 37°C. Subsequently, the organoids were quenched and treated briefly with DNAase I (Thermo Fisher Scientific, EN0521) and gently triturated using 2ml serological pipettes to generate single cell suspension in PBS with 1% BSA. Cell viability and count were assessed using trypan blue staining and a hemocytometer. The cell suspension was then loaded onto Chromium next GEM Chip G (10x Genomics, 2000177) per manufacturer instructions, with a targeted cell recovery of 5000-10000 cells. Post GEM formation, the samples were subjected to reverse transcription, amplification, fragmentation and clean-ups, as recommended by the manufacturer. We then performed Illumina library preparation using the Dual Index plate TT Set A (10X Genomics, 3000431; Plate, 15535894). The subsequent library was then sequenced using NovaSeq6000 at a depth of 25000-30000 paired end reads per cell.