GSM7519638: Renal LEC, saline, scRNAseq; Mus musculus; RNA-Seq
Kidneys were minced into small pieces and placed into 5mL digestion buffer (15mg/mL Collagenase D (Roche Applied Science, 11088858001), 10mg/mL Collagenase Type 4 (Gibco, 17-105-019), 2 mg/mL Dispase II (Roche Applied Biosciences, 4942078001), BSA Fraction V (Fisher Scientific, BP1605-100), and HBSS (ThermoFisher Scientific, 14025092). The samples were then incubated in a 37°C water bath with manual agitation every 10 minutes. Digestion was halted by adding 10mL of ice cold HBSS. Digested samples were filtered through both 70µm and 40 µm filters. Endothelial cells were enriched magnetically using a mouse CD31 biotinylated antibody (BD Pharmingen, 553371) and a cleavable Biotin Binder Kit (Invitrogen, 11533D). After the linker was cleaved, the isolated cells were subsequently enriched using an anti-mouse podoplanin (Pdpn) biotinylated antibody (BAF3244, R&D Systems) as above. CD31+Pdpn+ cells were placed into ice cold RPMI 1640 + 10% FBS prior to FACS confirmation of CD45-CD31+Pdpn+ cells. Cell fractions were resuspended in in RPMI1640 +10% FBS, counted, and diluted to a concentration of ~1,000 viable cells/μL. Library preparation was performed in accordance with the manufacturer's instructions. Renal LEC samples were mixed with master mix, paritioning oil, and gel then loaded onto the Chromium X Controller using the Chromium Next GEM Single Cell 3' Reagent Kit V3.1. Sample cDNA quality was checked using the Agilent High Sensitivity D5000 ScreenTape assay on the Agilent 4200 TapeStation System (Agilent Technologies, G2991BA). cDNA samples were then quantified and normalized prior to the library sequencing. Libraries were sequenced on a NovaSeq S4 Flow cell according to 10x Genomics recommendations. scRNA-seq. Libraries were sequenced on a NovaSeq S4 Flow cell according to 10x Genomics recommendations.