GSM5388412: Donor 2_RNA-seq; Homo sapiens; RNA-Seq
Human lung was micro-dissected under a dissection microscope to enrich for airways measuring less than 1 mm in their outer diameter and contiguous alveoli. The dissected pieces were placed in a dish containing DMEM/F-12 medium on ice and were longitudinally cut open. Samples were then dissociated in two steps. Step 1: samples were incubated in an enzyme cocktail containing 4 mg/ml pronase, 5 U/ml dispase, 1 mg/ml collagenease type IV and  0.33 U/ml DNase I  for 15 min at 37°C followed by rigorous pipetting for 1 min followed by another round of incubation for 15 min at 37°C. After incubation cells were centrifuged at 550 g for 10 min, supernatant was collected in a fresh 50 ml conical tube. Step 2: The cell pellet from step 1 was incubated 1 ml with TrypLE™ Select Enzyme (Thermofisher, #12563029) at 37°C for 10 min followed by rigorous pipetting for 1 min followed by another round of incubation for 10 min at 37°C. The cells were filtered through a 100 µm strainer and rinsed with 15 ml DMEM/F-12+10% FBS medium. The supernatant from step 1 and step 2 were combined and centrifuged at 550 g for 10 min. The supernatant was removed and the cell pellet was resuspended in red blood cell lysis buffer (Millipore Sigma, #11814389001 ) for 5 min, washed with DMEM/F-12 containing 10% FBS and filtered through a 40 µm strainer. Total cells were centrifuged at 550 g for 10 min at 4ºC. Cells were counted manually using Fuchs-Rosenthal hemocytometer (Incyto # DHC-F01). Followed the protocol of 10x Genmics Chromium v3.1 chemistry for single-cell RNA-seq (3' enrichment)