title: GSM4367152: In vivo, AAV-RSPO1-Fc D4; Mus musculus; RNA-Seq
library_construction_protocol: Intestinal adenomas of ApcMin/+ mice injected with AAV-RSPO1-Fc or empty vector were collected and incubated in a tissue dissociation solution (collagenase I 1 mg/mL, collagenase H 1 mg/mL, dispase II 4 mg/mL, DNase 4 mg/mL) for 30 min at +32°C. The suspension was incubated with Trypsin-EDTA (0.25%, phenol red, 25200056, Thermo Fisher Scientific) for 15 min at +37°C, gently pipetted to obtain a single-cell suspension and transferred into 0.04% BSA-HBSS. 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: 37 547 (ApcMin/+ in vivo D1-2 Ctrl), 47 141 (ApcMin/+ in vivo D1 RSPO1), and 38 871 (ApcMin/+ in vivo D2 RSPO1), 105 926 (ApcMin/+ in vivo D4 Ctrl), and 85 650 (ApcMin/+ in vivo D4 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.