title: GSM3307714: N703_SL273947; Mus musculus; ATAC-seq
library_construction_protocol: Native Renin cells were isolated from the kidney cortex of adult animals using FACS sorting, utilizing our Ren1c-YFP reporter mice. Ren1c-YFP and Ren1c-/-; Ren1c-YFP mice were anesthetized with tribromoethanol and the kidneys were excised, decapsulated, demedullated. The kidney cortices from each animal were dissected and minced to a smooth consistency, and transferred into a 15 ml Falcon tube with 5 ml of enzymatic solution [0.3% collagenase A (Roche, Indianapolis, IN), 0.25% trypsin (Sigma- Aldrich, St. Louis, MO), and 0.0021% DNase I (Roche Applied Science)]. The tubes were placed flat inside a shaking incubator (80 RPM) for 15 minutes at 37℃. Then, the solution was pipetted up/down ten times with a sterile transfer pipette and allowed to settle for 2 minutes, and the supernatant was collected with a plastic pipette and transferred to a fresh 15 ml tube on ice. The supernatants collected from the three digestions round were pooled and centrifuged in a Sorvall RT 6000 Refrigerated Centrifuge at 1,200 g for 4 minutes at 4°C. The cell pellet was resuspended with 25 ml of fresh buffer 1 (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM glucose, 20 mM sucrose, 10 mM HEPES, pH 7.4), and the suspension was poured through a sterile 100 µm nylon cell strainer (Falcon™ Cell Strainers, Corning, Inc. 352360) and washed with an additional 3–5 ml of buffer 1. The flow-through was poured through a sterile 40 µm nylon cell strainer (Falcon™ Cell Strainers, Corning, Inc. 352340) and washed with 3–5 ml of buffer 1. The flow-through was centrifuged at 1,200 g for 4 min at 4°C. After the centrifugation, the cell pellet was resuspended in 1ml of resuspension buffer [PBS, 1% FBS, 1 mM EDTA, DNAase I (Roche Applied Science)] and analyzed using a fluorescent-activated cell sorter (Influx Cell Sorter). We collected 50,000 cells for As4.1 and from chronically recruited mice, and 17,000 juxtaglomerular cells and cells from acutely recruited mice. Cells were washed with PBS and treated with Lysis Buffer. Then, cells were re-suspended in a transposition reaction mix (Illumina, cat # FC-121-1030) followed by DNA purification, before PCR amplification with 12-16 cycles with barcode primers (Illumina, cat # FC-121-1011). PCR products were purified with AmpureXP beads (Beckman Coulter, cat # A63881)
attributes.tag: GEO Accession
attributes.value: GSM3307714