title: Sample 1868
organism: Mus musculus
description: Protocols: The mouse stomach corpus was dissected and the tissue was dissociated into single-cell suspension by the incubation with Dispase ‖ (45U.ml). Then the single cells were sorted into 384 well plates containing lysis buffer by BD INFLUX. Spike-in RNA from the External RNA Controls Consortium (ERCC) (Ambion, Life Technologies) was added to each well in 1:640,000 dilution. Stomach cell sorting was performed by the WT Sanger Institute Flow Cytometry Facility for single cell RNA-seq data generation. 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 solutions 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. The supernatant was removed and 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 Stmn1+ cells by eGFP or Pgc+ cells by dsRed depending of the target cells we want to isolate in different single cell data generation. 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. 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-05-20T04:02:35Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-06-13T15:01:28Z
attributes.tag: External Id
attributes.value: SAMEA4727414
attributes.tag: INSDC center name
attributes.value: WT-MRC Cambridge Stem Cell Institute, The University of Cambridge
attributes.tag: INSDC first public
attributes.value: 2019-05-20T04:02:35Z
attributes.tag: INSDC last update
attributes.value: 2018-06-13T15:01:28Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6879:Sample 1868
attributes.tag: age
attributes.value: 9
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: epithelial cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: genotype
attributes.value: Pgc-IRES-DTR-T2A-dsRed
attributes.tag: individual
attributes.value: 21267
attributes.tag: organism part
attributes.value: stomach
attributes.tag: phenotype
attributes.value: Pgc positive
attributes.tag: post analysis well quality
attributes.value: fail
attributes.tag: sample name
attributes.value: E-MTAB-6879:Sample 1868
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: female
attributes.tag: single cell identifier
attributes.value: 21267_332
attributes.tag: single cell well quality
attributes.value: OK
attributes.tag: strain
attributes.value: C57BL/6