title: GSM6170833: Normal-OCTOPUS-d14, replicate 1, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: Enteroid lines are generated from terminal ileum mucosal biopsies. Briefly, two biopsy tissue fragments were rinsed 3 times in 1 ml cold sterile PBS, then incubated in cold chelation buffer for 30 minutes on a turntable in a cold room, followed by mechanical dis-sociation (scraping) of epithelial layer. The fragments were strained through a 100 μm strainer to deplete the villi and resuspended in 80% Matrigel, then seeded at the density of 50-200 crypts per 30 μl drop. The droplets solidified at 37ºC for 30 minutes, and 500 μl human IntestiCult (STEMCELL Technologies; complete when supplemented with Penicillin-Streptomycin (Gibco)) was added per well. Y-27632 (SelleckChem; final conc. 10 μM) was added to culture medium at seeding only. Single-cell suspension for each organoid sample was loaded onto a separate channel of a Chromium 10X Genomics Single Cell 3' Reagent Kit v2 library chip (10X Genomics) according to the manufacturer's protocol. RNA transcripts from single cells were uniquely barcoded and reverse-transcribed. cDNA sequencing libraries were prepared according to the manufacturer's protocol (10X user guide for library prep) and sequenced on an Illumina NovaSeq 6000 using an S1 100 cycles flow cell v1.5. Library quality control was done using Agilent TapeStation for sizing (bp) and KAPA qPCR for concentration (nM).