Heterocephalus glaber
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. 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).
isolate
not applicable
ENA-FIRST-PUBLIC
2023-08-19T00:36:18Z
individual
OE0538_DO-0001_hgla_AC_7w_1
organism
Heterocephalus glaber
organism
Heterocephalus glaber
ENA-LAST-UPDATE
2023-08-19T00:36:18Z
sex
male
scientific_name
Heterocephalus glaber
common name
naked mole-rat
organism part
skin of body
age
17
developmental stage
adult
genotype
wild type genotype
ENA-FIRST-PUBLIC
2023-08-19
ENA-LAST-UPDATE
2023-08-19