title: GSM6424658: PAH_1, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: Pulmonary arteries were dissected under stereomicroscope by following the branching pattern and including accompanying perivascular (adventitial) region. Arteries were minced and digested for 60 min at 37°C in phosphate-buffered saline (PBS) containing 2 mg/mL collagenase A (Roche) and DNase I (Sigma). Digest was filtered through 100 micron cell strainer into FBS and diluted with PBS. Following centrifugation, cell pellet was washed with PBS and passed through 40 micron cell strainer. The secondary, and tertiary pulmonary arteries from the left lung lobe were isolated from the lungs of 12 week old male mice kept under normobaric hypoxia (10% O2) or normoxia (21% O2) for three weeks minced and digested as described for the human counterpart. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.