Illumina HiSeq 2500 sequencing; Single cell RNA-seq of Pgc+ cells in homeostasis and upon injury
Single cell RNA-seq of Pgc+ cells in homeostasis and upon injury
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. After dissociation, the cell pellet was resuspended in 100 µL of antibody mix (1% FBS; 10 U/mL DNAse, Promega; 1:125 Alexa Fluor® 647-conjugated anti-mouse/human CD324 (E-Cadherin) antibody, BioLegend) and incubated for 1 h on ice. Cells were washed with 3 mL 1% FBS in PBS and filtered once more if clumps could be observed. After centrifugation at 300 g for 5 min the cell pellet was resuspended in 1 mL 1% FBS and 10 U/mL DNAse in PBS for sorting. BD INFLUX systems were used for sorting in the WT Sanger Institute Flow Cytometry Facility. After the viable cells were gated at first, the cell were subject to the second gating to select only singlet cells. Then the third gating was to select E-Cadherin+ epithelial cells by Alexa 647. The fourth gating was performed to select Pgc+ cells by dsRed. Each well of the 384 well plate contained 2.3 ul of lysis buffer with RNAse inhibitor (Ambion) in a 0.2% (v/v) Triton X-100 solution.  Parietal cell depletion was performed by DMP-777 administration by oral gavage (5.6 mg per 20 g body weight) Preamplification was performed in a total volume of 27 ul that contained 13.5 ul of HiFi Hotstart ReadyMix (2x; KAPA Biosystems) and 0.1 uM of IS PCR primer (Sigma-Aldrich). After 25 cycles of amplification, samples were cleaned with 80% (v/v) of Ampure beads (Beckman Coulter). Sequencing libraries were prepared using the Nextera XT DNA sample preparation kit (Illumina).