title: GSM4773488: Coarctation_Mouse_3_Right; Mus musculus; RNA-Seq
library_construction_protocol: Renin cells were isolated from the kidney cortex of the Ren1c-YFP mice at 8 to 12 weeks of age after 72 hours of aortic coarctation or sham surgeries using FACS. A kidney cortex was dissected, minced with a razor blade, and transferred into a 15 mL tube with 5 mL of enzymatic solution (0.3% collagenase A [Millipore-Sigma], 0.25% trypsin [Millipore-Sigma], and 0.0021% DNase I [Millipore-Sigma]). The tubes were placed flat inside a shaking incubator (80 RPM) for 15 minutes at 37°C. 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 in a fresh tube on ice. The enzymatic solution was added to the 15 mL tube containing the remaining undigested cortexes, and the digestion procedure was repeated a total 3 times. The supernatants collected from the 3 digestions were pooled and centrifuged at 1,100 g for 4 minutes at 4°C using a Sorvall RT7 refrigerated centrifuge (Sorvall, Newtown, CT). The cell pellet was resuspended with 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 (Corning Inc., Corning, NY) and washed with the buffer 1. The flow-through was poured through a sterile 40 μm nylon cell strainer (Corning Inc.) and washed with buffer 1. The flow-through was centrifuged at 1,100 g for 4 min at 4°C. The cell pellet was resuspended in 1.5 mL of resuspension buffer [PBS, 1% FBS, 1 mM EDTA, DNAase I (Millipore-Sigma)]. The dead cells were labeled with DAPI (Millipore-Sigma). Cells were analyzed and sorted by FACS using Influx Cell Sorter (Becton Dickinson, Franklin Lakes, NJ). The living cells labeled with YFP not staining positive with DAPI were sorted and directly collected into the 1x Reaction buffer of SMART-Seq v4 Ultra Low Input RNA Kit (Takara Bio USA, Mountain View, CA). One thousand cells from each kidney were used to make the cDNA library of each sample. Samples were prepared using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio USA, Mountain View), according to the user manual. The cDNA was amplified with 13 cycles of PCR. cDNA was isolated using the Agencourt AMPure XP Kit (Beckman Coulter, Brea, CA) and eluted in the Fluidigm dilution reagent (Takara Bio USA). Samples were quantified using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA) and qualified using the High Sensitivity D5000 ScreenTape System (Agilent Technologies, Santa Clara, CA). Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) was used to make cDNA libraries tagmented and indexed with unique barcodes suitable for Illumina sequencing.