title: GSM4365350: organoid, Ctrl; Mus musculus; RNA-Seq
library_construction_protocol: In order to obtain single cell solution, itestinal organoids derived from ApcMin/+ mice treated with or without RSPO1-Fc fusion protein were incubated in Hanks' balanced salt solution (HBSS, 14175-053, Gibco) containing 1 mg/mL collagenase type I (LS004196, Worthington), 1 mg/mL collagenase H (11074032001, Roche), 4 mg/mL dispase II (04942078001, Sigma) and 1000 U/mL benzonase (sc-202391, ChemCruz) for 30 min at 37°C with gentle shaking, followed by 10 min incubation with trypsin. Cells were transferred into 0.04% BSA-HBSS for analysis. Single cells were analyzed using the 10x Chromium Single-Cell 3′RNA-sequencing system (10x Genomics, Pleasanton, CA, USA) with the Reagent Kit v2 as instructed by the manufacturer. Briefly, the cells were first loaded into Chromium Single-Cell Chip v2 (10x Genomics, Pleasanton, CA, USA), followed by generation of Gel beads in EMulsion (GEM) aiming at 5000 cell captures per sample. This was followed by cDNA purification, amplification (12 cycles), and library construction (sample index PCR 14 cycles) as instructed by the manufacturer. The sample libraries were sequenced on an Illumina NovaSeq 6000 sequencer using read lengths of 26bp (Read 1), 8bp (i7 Index), 0 bp (i5 Index) and 91bp (Read 2),  resulting in an average of following reads per cell: 58 414 (Ctrl), and 131 291 (RSPO1). Chromium single-cell 3' RNA-sequencing barcodes and read data were demultiplexed and converted to FASTQ files, and aligned reads and gene-cell matrices were generated using Cell Ranger v 2.1.1 mkfastq and count pipelines (10x Genomics, Pleasanton, CA, USA). Reads were aligned to mouse reference genome mm10.