GSM6754177: Biol rep 2 - GFP-HI; Sus scrofa; RNA-Seq
Cells were isolated from lungs of adult or fetal LGR5 pigs. 2-3 cm2 pieces of lung were collected in cold PBS and moved into a sterile hood. Lung tissue was then minced using scissors or razor blade and then washed twice with cold PBS to remove blood and immune cells. Minced tissues were then incubated for 1 hour in a roller incubator at 37℃ in an enzymatic digest buffer containing 10 mg/mL Dispase (Invitrogen), 2 mg/mL DNase I (STEMCELL Technologies), and 5mg/mL (adult) or 2mg/mL (fetal) Collagenase Type II (Sigma). The digestion mixture was then strained using a 70 μM cell strainer and washed with a wash buffer (PBS, 5% FBS, 1% antibiotic-antimycotic) and centrifuged. For adult tissue, pellets were incubated in Red Blood Cell Lysis Buffer (Invitrogen) for 10 minutes at room temperature with gentle agitation. For fluorescent activated cell sorting, cells from porcine lung or dissociated organoids were suspended in MACS buffer (Miltenyi) and incubated with 10μl/ml anti-EpCAM-APC (Thermo Scientific, 17-5791-83) and/or anti-NGFR-PE (eBioscience, 12-9400-41). Propidium iodide was added for dead cell exclusion immediately before fluorescence activated cell sorting (Beckman Coulter MoFlo XPD). Illumina Nextera XT library was used for sequencing library preparation.