GSM8458657: Lung, Aged Non-Ventilated; Mus musculus; RNA-Seq
Mice were euthanized via intraperitoneal injection of pentobarbital sodium. The pulmonary vasculature was flushed with sterile PBS through the right ventricle of the heart. The lungs were removed and the left lobe was isolated and minced to small pieces (approximately 1-3mm2). The tissue pieces were enzymatically digested for 30 minutes in complete Roswell Park Memorial Institute media (RPMI) containing 10% fetal bovine serum and 1% penicillin/streptomycin, along with 400 µg/mL each of the digestion enzymes, LiberaseTM and DNaseI. The enzymatic reaction was then stopped using 100mM EDTA. Dissociated cells in suspension were passed through a 100μm cell strainer using ice-cold 2mM EDTA in complete Dulbecco's Modified Eagle Medium (DMEM supplemented with 10% FBS and pen-strep). Centrifugation was performed, and cells were resuspended in ice-cold DMEM with an aliquot being taken to perform cell counts. Centrifugation was performed again, followed by resuspension of the cells in ice-cold DMEM at a concentration of 1 million cells/mL. An additional aliquot was taken for cell counts and cell viability assessments using trypan blue. Centrifugation was performed one last time and cells were resuspended in CryoStor CS10 freezing media at 2 million cells/mL. Cells were transferred to an isopropanol cooling container and placed in a -80°C freezer overnight. The cells were then transferred the following day to a liquid nitrogen storage tank for long-term storage until all samples were collected. Each treatment group yielded n=3 animals pooled per condition. Mouse lung single cell suspensions were rapidly thawed from cryopreservation. Cell suspensions were washed with ice-cold PBS with 0.5% BSA then counted. Cells were resuspended in PBS with 0.5% BSA. Viability was assessed with trypan blue exclusion. Samples with a viability of less than 80% were excluded. Samples from each treatment group were proportionately pooled to a final count of 5000 cells for each treatment, which were subsequently used for library preparation. The Cells were prepared for cDNA amplification and chromium library construction using the 10x Chromium Next GEM Single Cell 3' Library and Gel Bead Kit v3.1 according to manufacturer's protocol (10x Genomics, Pleasanton, CA, USA).