single cell RNAseq (10x Genomics) of small mesenteric artery in Rattus norvegicus
Cell concentrations and viability were determined using the Cell Countess II FL (ThermoFisher) prior to calculating cell counts and loading the suspension into the Next GEM Chip G and Chromium Controller (10x Genomics) per the manufacturers protocol for GEM formation. The 10x Chromium Next GEM 3' protocol was used to create 3' libraries; briefly, cells were lysed and barcoded within each GEM prior to first strand cDNA synthesis, also within each GEM. After GEMs are broken, cells are pooled prior to library completion as described in the manufactures protocol. Library quality was assess after cDNA synthesis and after completion on the BioAnalyzer using a DNA High Sensitivity Chip (Agilent). Prior to sequencing, Agilent Tape Station 4200 and Invitrogen Qubit 4.0 reagents were used determine final library concentrations prior to dilution, normalization and pooling at 4nM. qPCR was used to determine cluster efficiency.
SM07