GSM8919863: Cortex_3D_ANGII_CA_42D3_BiolRep3; Mus musculus; RNA-Seq
A total of 12 cell suspensions were generated, with 3 biologic replicates per treatment group. Within each replicate, 2 cortical hemispheres were pooled from 2 identically treated mice. Mice were anesthetized by isoflurane inhalation (5% induction, 2% maintenance) and perfused transcardially with ice-cold sucrose-HEPES “Cutting Buffer” containing (in mM) 110 NaCl, 2.5 KCl, 10 HEPES, 7.5 MgCl2, 25 glucose, and 75 sucrose (~350 mOsm kg−1). The brain was removed, placed in ice-cold Cutting Buffer, then transferred to a slicing chamber filled with ice-cold Cutting Buffer. 400 μm thick brain slabs were cut with a vibrating microtome. Slabs containing the regions of interest were gently transferred to a dissection dish with ice-cold dissociation buffer (DB) containing (in mM): 82 Na2SO4, 30 K2SO4, 10 HEPES, 10 glucose and 5 MgCl2. Regions of interest were isolated, then transferred into a 15 ml falcon tube filled with 5 ml of DB + enzyme buffer (DEB) consisting of DB with 3 mg/ml of Protease XXIII (Sigma-Aldrich, P5380), 10 units/ml of Papain (Sigma-Aldrich, P4762), 0.5 mM L-Cysteine (Sigma-Aldrich, 168149), and 0.25 mM EDTA (Worthington, LK003153). Digestion was performed at 34 °C for 2 hours. Tubes containing digested tissue were transferred onto ice and the DEB was replaced with 10 ml of a DB + stop solution (DBS) containing DB and 1 mg/ml Trypsin Inhibitor (Sigma-Aldrich, T6522), 2 mg/ml BSA (Sigma-Aldrich, A2153), and 1 mg/ml Ovomucoid Protease Inhibitor (Worthington, LK003153). Digested tissues were then dissociated using a series of 4 fire-polished Pasteur pipets with successively smaller bores. Bubbles were avoided. Falcon tubes containing 10 ml of dissociated cells were then centrifuged at 300 g for 10 minutes. The supernatant was removed and discarded, taking care not to disturb the cell pellet. The pellet was resuspended in 5 ml of DBS and centrifuged again under the same conditions. The supernatant was once again removed, and the cleaned cell pellet was resuspended to a volume of 3 ml in DB containing 0.01% BSA (w/v). Suspensions were passed into a new tube through a pre-wet 40 μm filter and held on ice. 2 samples of 10 μl were drawn from the suspension and mixed 1:1 with 10 μl of 2× dye mix containing DB and 20 μM EthD-1 (Thermo Fisher Scientific, L-3224), 20 μM Calcein-AM (Thermo Fisher Scientific, L-3224), and 40 μM Hoechst 33342 (Thermo Fisher Scientific, 62249). After 5 minutes of incubation at room temperature, 10 μl from each sample were loaded onto a haemocytometer and imaged using a fluorescent Olympus IX83 microscope. For each of the 2 samples, three random locations were imaged using DIC and three fluorescent channels to capture the dyes. These images were used to calculate cell concentrations and metrics of cell intactness. Afterward, cells were resuspended in 0.01% BSA-PBS at a concentration of 100,000 cells/ml for Drop-seq processing. Single-cell transcriptomes of cortical brain cells were prepared by Drop-seq. Cells were resuspended in 0.01% BSA-PBS to a final concentration of 100 cells/μl. Barcoded capture beads (ChemGenes Corporation, Wilmington, MA) were resuspended in 1.8 ml lysis buffer consisting of 4M Guanidine HCL (Thermofisher Scientific, 24110), 6% Ficoll PM-400 (Sigma-Aldrich, F4375), 0.2% Sarkosyl (Sigma-Aldrich, 61747), 20mM EDTA (Thermofisher Scientific, 17892), 200mM Tris pH 7.5 (Sigma-Aldrich, DB0339), and 50mM DTT (Sigma-Aldrich, D0632) at a concentration of 120 beads/μl. A 5 mm diameter, 1.7 mm thick PVDF encapsulated magnetic stir disc and rotary magnetic tumble stirrer (V&P Scientific, VP772DP-N42-5-2) was used along with a 3 ml syringe that contained the beads in lysis buffer to maintain the beads in suspension. Next, single cells and beads were encapsulated in nanoliter-scale droplets using a Drop-seq microfluidic device coated with Aquapel (FlowJEM); flow rates were 15 ml/hour for the Droplet generation oil (BioRad, 1863005) and 4 ml/hour for the cells and beads. Each run lasted approximately 18 minutes. After removing the oil, droplets were resuspended in 30 ml of room temperature 6X SSC (Promega, V4261) and 1 ml perfluorooctanol (Sigma-Aldrich, 370533) and shaken vigorously 6 times vertically to break the droplets. Beads were then captured by loading them into a 20 ml syringe with an attached 0.22 μm Millex-Gv syringe filter (Millipore Sigma, SLGV033R). Beads were washed with 2 x 20 ml of ice cold 6X SCC. After washing, the syringe filter was inverted and flushed 3x with 10 ml of ice cold 6x SSC using a 10 ml syringe. Beads were collected from the resultant solution via centrifugation at 1,250 x g for 2 min at 4°C with low brake setting. cDNA was amplified by PCR using the following parameters: 95 °C (3 min); 4 cycles of 98 °C (20 s), 65 °C (45 s), and 72 °C (3 min); 11 cycles of 98 °C (20 s), 67 °C (20 s), and 72 °C (3 min). Libraries were quantified by quantitative PCR and checked for quality and size distribution on a Bioanalyzer (Agilent) before sequencing on an Illumina NextSeq500 instrument using the 75 cycle High Output v2 kit (Genomics Core Facility, Cornell University, Ithaca, NY). Three to four libraries were multiplexed into a single run. We loaded a 1.8 pM library and provided Drop-seq Custom Read1 Primer at 0.3 μM in position 7 of the reagent cartridge without PhiX spike-in using a read configuration of 20 bases (Read1), 8 bases (Index1), and 64 bases (Read2).