title: Illumina HiSeq 4000 sequencing; Single cell sequencing of renal endothelial cell heterogeneity and metabolic transcriptome adaptation to dehydration.
design: Single cell sequencing of renal endothelial cell heterogeneity and metabolic transcriptome adaptation to dehydration.
library_construction_protocol: Mice were exposed to food and water ad libitum, or water-deprived, for 12h, 24h, 36h or 48h (n=6/group). Mice were anesthetized with an intraperitoneal injection of Ketamine/Xylazine and blood was collected from the right ventricle. Mice were then submitted to left heart perfusion with 3mL PBS (1ml/min) and perfused kidneys were collected in ice-cold HBSS. The 60 kidneys were collected the same day to avoid batch effects. Kidney capsules were removed and kidneys were further dissected under microscope to separate cortex from medulla. Kidney cortices and medullae from each condition were then cut into small pieces in a collagenase digestion buffer (2mg/mL collagenase I (C9891, Sigma), 27IU/mL DNAse I (D4527, Sigma) and HBSS (14025100, Thermofisher scientific)). 15ml were used for cortex samples, and 5ml for medulla samples. Kidney tissues were then digested 40 min at 37°C, shaking every 10 min, and stopped adding 20ml ice-cold HBSS. Digested cortex and medulla samples were sequentially filtered in 70µm and 40µm filters and glomeruli were collected in the 40µm filter from cortex samples and resuspended in HBSS. RECs from cortex and medulla were enriched using mouse CD31 magnetic microbeads (130-097-418, Miltenyi Biotec) and QuadroMACS separators with LS columns (Milteniy Biotec). Collected glomeruli were further dissociated in a trypsin digestion buffer (0.2% trypsin (25200056, Thermofisher scientific), 100IU/mL DNAse I, 100µg/mL Heparin (Heparine LEO, LeoPharma)), 23 min, at 37°C. Trypsin was inactivated adding 10% FBS in ice-cold HBSS. Glomerular cells were centrifuged 6 min, at 400g, and resuspended in wash buffer (0.1% BSA, PBS). Cortical and medullary endothelial-enriched cells as well as glomerular cells were incubated with CD102-Alexa647 antibody (1/300, A15452, Thermofisher scientific), CD45-PeCy7 antibody (1/500, 130-097-418, Thermofisher scientific), CD73-PerCP-Cy5.5 antibody (1/100, 127213, Biolegend, for glomerular cells only) and viability dye eFluor450 (1/1000, 65-0863-14, Thermofisher scientific) in FACS buffer (0.1% BSA, 2mM EDTA in PBS) for 30 min at 4°C. OneComp ebeadsTM (01-1111-42, Thermofisher Scientific) and cooked cells (20 min, 60°C) were used for compensation. 100,000 viable ECs were FACS-sorted, using FACS Aria III, and collected in wash buffer (0.1% BSA, PBS). Cells were centrifuged 6 min at 400g, and resuspended in 40µL wash buffer. Cell viability and number was assessed staining cells with acridine orange and propidium iodide (F23001, Logos Biosystems) and using the automatic dual fluorescence cell counter system (Luna-Fl, Logos Biosystems).  Nucleic acid extraction was done in single cells using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (v2, 10x Genomics) according to manufacturer's instructions.  The single cell suspensions of FACS-sorted glomerular renal ECs (gRECs), cortical renal ECs (cRECs) and medullary renal ECs (mRECs) were converted to barcoded scRNA-seq libraries using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (10x Genomics; Pleasanton, CA, USA, PN-120237, PN-120236), aiming for approximately 4,000 cells per library of control gRECs, cRECs and mRECs and 3,000 cells of RECs from each compartment and for each dehydration timepoint. Samples were processed with 10x Genomics' V2 barcoding chemistry. Cells from each sample were treated with the same master mix and in the same reaction vessel, by processing every single sample in a single well of a PCR plate. All samples were processed in parallel in the same thermal cycler.