GSM9285307: Rat lung - healthy and induced PAH model at 1, 3, 5, and 8 weeks, pool B2, LMO; Rattus norvegicus; RNA-Seq
Animals were perfused with 20ml of 0.9% saline containing 25U/ml heparin (LEO Pharm Inc.) through  the right ventricle, to drain the blood from the lungs. Lung tissue was isolated, minced with scissors and placed in Hank's Balanced Salt Solution (HBSS, Gibco) on ice. Enzymatic digestion was performed with 2500U Collagenase (Worthington), 30U Neutral Protease (Worthington), and 165U DNase I (Sigma Aldrich) in HBSS, samples were loaded into gentleMACS C tubes (Milltenyi Biotec), and placed on the OctoMACS dissociators (Milltenyi Biotec) using program 37C_LDK_m_1 for ~30min. Samples were immediately placed on ice, filtered through 70µm cell strainers (Falcon), and washed with phosphate buffered saline (PBS, Gibco) with 2mM EDTA (ThermoFischer Scientific) and 0.5% bovine serum albumin (BSA, Wisent) (PEB). Dissociated cell samples were spun at 400g for 5min at 4°C and washed again. Samples were incubated in 1x red blood cell lysis (eBioscience) for 3min, diluted with PEB, and filtered again through 70µm cell strainers (Milltenyi Biotec). Individual biological samples were barcoded using the MULTI-seq protocol described by McGinnis et al. In brief, single cells were incubated with 1:1 molar ratio of anchor: unique barcode oligonucleotide sequence for 10 min at room temperature with gentle mixing. Followed by addition of the co-anchor to stabilize barcodes within membranes which was incubated for 5min on ice. Barcoded cells were washed in PBS, counted as above, and pooled at an equal ratio of cells per biological replicates. Two aliquots from the pooled library preparation were run on separate channels of the 10X chromium controller.