characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2022-05-05T00:15:53Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2022-05-05T00:15:53Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA13657541
characteristics.characteristic: External Id
characteristics.text: Department of Life Sciences, POSTECH
characteristics.characteristic: INSDC center name
characteristics.text: 2022-05-05T00:15:53Z
characteristics.characteristic: INSDC first public
characteristics.text: 2022-05-05T00:15:53Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS11259359
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-11587:Sample 2
characteristics.characteristic: Submitter Id
characteristics.text: 12
characteristics.unit: week
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: stomach epithelial cell
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Stomachs were prepared by carefully separating the corpus from the forestomach and pylorus. Corpus tissue was divided into four 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 of 10% FBS in DMEM 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 scRNA-seq for Gif lineage cells, we utilized Gif-rtTA; TetO-Cre; Rosa-nTnG mice as described above. We administrated doxycycline by drinking water as described for animal treatments. Stomach samples from uninjured or DMP-777-treated mice at 2 dpi were collected separately and cells were dissociated. To sort GFP+ DsRed- cells only, we used WT stomach cells as a control to identify GFP+ DsRed+ cell windows in the FACS plot. The cells were sorted in 384-well plates. Plates were immediately frozen at -80 °C until they were shipped in dry ice to Single Cell Discoveries. Cells were lysed by 5 min at 65°C, after which RT and second strand mixes were dispersed with the Nanodrop II liquid handling platform. Aqueous phase was separated from the oil phase after pooling all cells in one library, followed by IVT transcription. The CEL-Seq2 protocol was used for library prep. Primers consisted of a 24 bp polyT stretch, a 4bp random molecular barcode (UMI), a cell-specific 8bp barcode, the 5′ Illumina TruSeq small RNA kit adaptor and a T7 promoter. mRNA of each cell was then reverse transcribed, converted to double-stranded cDNA, pooled and in vitro transcribed for linear following the CEL-Seq 2 protocol (Hashimshony et al., 2016).
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: Gif-rtTA; TetO-Cre; Rosa-mTmG
characteristics.characteristic: genotype
characteristics.text: TdTom- GFP+
characteristics.characteristic: immunophenotype
characteristics.text: s005
characteristics.characteristic: individual
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: stomach
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.characteristic: sex
characteristics.text: doxycycline
characteristics.characteristic: stimulus
characteristics.text: Sample 2
characteristics.characteristic: title