title: Illumina HiSeq 2500 sequencing; Time course bulk RNA-seq of Troy+ chief cells in the mouse stomach corpus epithelium after injury
library_construction_protocol: Stomachs were prepared by carefully separating the corpus from the forestomach and pylorus. Corpus tissue was divided into 4 pieces of similar size and incubated in 4 ml dissociation solution (45 U/ml Dispase II, Thermo Fisher Scientific; 0.6 mg/ml pancreatin, Sigma; 1x penicillin/streptomycin in DMEM high glucose, HEPES, no phenol red, Thermo Fisher Scientific) at 37 °C with shaking at 270 rpm. After dissociation, the solution becomes cloudy and corpus fragments appear more transparent. All subsequent steps were then performed on ice. To avoid cell loss due to cell adhesion to pipette or tube walls, all pipettes and tubes used for pipetting the cell suspension were pre-incubated with either DMEM or PBS containing FBS. To further disrupt the tissue, the cell suspension, including corpus pieces, was pipetted up and down several times using a 10 ml pipette. The cell suspension without remaining corpus pieces was then transferred to a 15 ml Falcon tube. The corpus pieces and the dissociation tube were then washed twice with 5 ml DMEM + 10% FBS and the wash solution combined with the cell suspension for inactivation of the dissociation reaction. Cells were centrifuged at 300 g for 5 min and resuspended in 1% FBS in PBS. The cell suspension was filtered through a 100 μm cell strainer into a pre-coated 15 ml tube. The tube and filters were washed twice with 1 ml of 1% FBS in PBS and the cell suspension was then centrifuged a second time at 300 g for 5 min. For Troy+ chief cell sorting, supernatant was removed and the cell pellet was resuspended in 100 μl antibody mix (1% FBS, 10 U / ml DNAse; Promega, 1 to 20 ATPase, H+ / K+ Exchanging, beta polypeptide (ATP4b) Alexa Fluor 555 rabbit antibody; Bioss) for 1 h incubation on ice. Cells were washed with 3 ml PBS + 1% FBS and filtered once more if clumps could be observed. After 5 min centrifugation at 300 g, the cell pellet was resuspended in 1 ml PBS, 1% FBS, 10 U / ml DNAse and transferred to MOFLO tube for sorting. Cells were sorted directly into 300ul lysis buffer (Arcturus Pico Pure RNA Isolation Kit). Cell lysate was snap-frozen on dry ice and subsequently stored at -80 ºC. RNA purification was performed using the Arcturus Pico Pure RNA Isolation Kit (Life Technologies) following the manufacturer's instructions. For single-end mRNA-sequencing, 17 total RNA samples were extracted from Troy+-eGFP gastric corpus cells in homeostatic and injury conditions. The integrity of total RNA was confirmed using an Agilent BioAnalyzer. The ranges of RNA integrity number (RIN) were 6.6~9.3. cDNA libraries were generated from total RNA according to QuantSeq 3' mRNA-Seq FWD library preparation protocols. The library templates were amplified in 14 -18 PCR cycles, and concurrently barcoded. Purification was performed with 27 μl PB, and the resulting end-point PCR products were analysed on a bioanalyser using a DNA HS chip. 6 - 10 fmol of each library (calculated from the 150 - 1500 nt region) were pooled to yield an equimolar lane mix. Each lane contained libraries from 12 or 13 samples. The lane-mixes were separately subject to single end 100 nt sequencing using an Illumina HiSeq 2500 system.