characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-11-21T00:50:46Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-11-21T00:50:46Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112302679
characteristics.characteristic: External Id
characteristics.text: Junior Research Group Somatic Evolution and Early Detection German Cancer Research Center (DKFZ), Heidelberg, Germany
characteristics.characteristic: INSDC center name
characteristics.text: 2023-11-21T00:50:46Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-11-21T00:50:46Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14413611
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12451:sc-exp_004_skin-9097-2
characteristics.characteristic: Submitter Id
characteristics.text: 90
characteristics.unit: week
characteristics.characteristic: age
characteristics.text: sc-exp_004_skin-9097-2
characteristics.characteristic: biopsy name
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: naked mole-rat
characteristics.characteristic: common name
characteristics.text: Protocols: Naked mole rats (Heterocephalus glaber) or mice (Mus musculus, C56BL6/J) were culled by neck decapitation and full-thickness 4 and 1 mm2 dorsal skin biopsies, respectively, were immediately collected. In brief, shaved backs of female mice were treated once with 100 ug DMBA (Sigma-Aldrich) in 100 ul acetone. After one week, 10 ug TPA (Sigma-Aldrich) in 100 ul acetone was applied three times a week. The mice were sacrificed at the first macroscopic occurrence of papillomas (weeks 10-12). Male naked mole-rats were treated once with 500 ug DMBA (Sigma-Aldrich) in 250 ul acetone. After one week, 60 ug TPA (Sigma-Aldrich) in 250 ul acetone was applied three times a week for 12 or up to 24 weeks. Skin biopsies were incubated in dissociation buffer containing 1.25 mg/ml Collagenase I (Sigma), 0.5 mg/ml Collagenase II (Worthington), 0.5 mg/ml Collagenase IV (Worthington) and 0.1 mg/ml Hyaluronic acid (Sigma) for 3 hours at 37°C, 750 rpm. Enzymes were diluted in a final volume of 0.5 ml Hanks Balanced Salt solution (HBSS) per sample (Invitrogen). After 3 hours 0.5 ml 0.5 % Trypsin (Gibco) was added (to a final concentration 0.25%) and incubated at 37C for an additional 30 min at 750 rpm. The Enzymatic digestion was quenched with HBSS supplemented with 10% fetal calf serum (FCS, Gibco) and the cell suspensions were filtered through 40-µm cell strainers (Grenier Bio-One). The strainers were washed with an additional 2-3 ml HBSS containing 10% FCS and cells pelleted by centrifugation at 300 x g for 8 min at 4°C. Cell pellets were then carefully (using wide-bored P1000 pipet tips) resuspended in 100-200 ul sorting buffer (PBS containing 1 mM EDTA (Invitrogen) and 0.04% BSA (Sigma) and 1 μg/ml propidium iodide (Invitrogen)). Cell suspensions were carefully transferred to low-binding polypropylene FACS tubes. Live cells from each sample were sorted into one 96-well flat-bottom low attachment tissue culture plate well containing 30 ul sorting buffer on a cold plate on an BD FACS Aria 2 Len sorter using a 70 µm nozzle (70 psi). PI was excited with a 561 nm laser and fluorescence was recorded with a 610/20 595LP filter. Cells were gated i) FSC-A vs SSC-A to remove debris ii) SSC-W vs SSC-A to exclude doublets iii) RL780/60-A vs PI-A to exclude auto-fluorescent cells and dead cells (PI positive). Single cell cDNA libraries were prepared using the 10X Genomics Chromium Single Cell 3’ kit (User Guide Version 3) according to the manufacture’s instruction. Quantification of the libraries was carried out using the Qubit dsDNA HS Assay Kit (Life Technologies), and cDNA integrity was assessed using D1000 ScreenTapes (Agilent Technologies).
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: 9097
characteristics.characteristic: individual
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Heterocephalus glaber
characteristics.characteristic: organism
characteristics.text: Heterocephalus glaber
characteristics.characteristic: organism
characteristics.text: skin of body
characteristics.characteristic: organism part
characteristics.text: Heterocephalus glaber
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.characteristic: sex
characteristics.text: Cambrigde/McGonagall
characteristics.characteristic: strain
characteristics.text: sc-exp_004_skin-9097-2
characteristics.characteristic: title
characteristics.text: to
characteristics.characteristic: treatment