title: GSM6369018: scRNA-seq from Ramp1 control colon; Mus musculus; RNA-Seq
library_construction_protocol: Briefly, distal colon was collected from euthanized mice and flushed with ice cold PBS. Colons were cut into 0.5cm pieces and incubated on ice for 5 min in 10 mL of PBS containing 5 mM EDTA and 1 mM DTT. After shaking, the intestinal pieces were incubated at 37 ºC for 10 minutes in PBS containing 5 mM EDTA and 2% FBS. After vigorous shaking, the wash containing crypts was collected. This step was performed three times in total for colonic pieces and two times for ileal pieces with the epithelial fraction collected at the end of each wash. The crypt cells were pelleted and digested with 10 ml HBSS containing 8 mg Dispase II (Sigma) for 10 minutes at 37 ºC. Single epithelial cells were collected in PBS containing 2% FBS for flow cytometry staining and sorting. scRNA-seq of colonic epithelial cells isolated from Nav1.8DTA mice, VillinCreRamp1 knockout mice and their littermate control mice was performed using 10X genomics following the user guide. Briefly, cells were counted on the LUNA-FX7 Automated Cell Counter (Logos Biosystems) using fluorescence detection for viability with an acridine orange/propidium iodide stain (Logos Biosystems). All samples had viability greater than 88% with concentration ranges from ~700-900 cells/µL. After counting, all samples were loaded into Chip G per the user guide from 10x Genomics. GEMs (Gel Beads-in-emulsions) were formed targeting 10,000 cells for each sample followed by immediate reverse transcription. The cDNA was cleaned from the GEM reagents, amplified for a total of 11 cycles and verified via TapeStation (Agilent Technologies). Amplified cDNA was diluted 1:6 and run on the 4200 TapeStation instrument using High Sensitivity D5000 tape and reagents (Agilent Technologies). The amplified cDNA was fragmented, end repaired, and A-tailed followed by adaptor ligation, and PCR amplification was run for a total of 11 cycles with each sample receiving a unique set of dual indices. Final libraries were diluted 1:10 and ran using the High Sensitivity D5000 tape and reagents on the 4200 TapeStation (Agilent Technologies). Libraries were quantified via Kapa qPCR using the Complete Universal Kit (Roche Sequencing Solutions) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories). Libraries were sequenced on an Illumina NovaSeq S4 flow cell across one single lane (estimated reads per cell ~55,000) using the parameters outlined in the user guide (Read1: 28 bp, i7 index: 10 bp, i5 index: 10 bp, Read2: 90 bp).