GSM4932752: Hypoxia1; Mus musculus; RNA-Seq
CAG-CreERT2-ODD::R26R-tdTomato mice, 4 weeks old, received tamoxifen (100mg/kg/d) daily for 5 consecutive days before being exposed to either inspiratory hypoxia (6%O2) or normoxia (21%O2) for 6h each. Mice were handled identically and sacrificed on day 5 immediately after the last hypoxia/normoxia session. Hippocampi were dissected in Earle's Balanced Salt Solution (EBSS1) and later digested with a working solution of Papain/DNaseI prepared in EBSS1, according to manufacturer's instructions (Worthington Biochemical Corp). All steps were carried out on ice except the Papain/DNaseI dissociation. The samples were then incubated at 37°C for 40min with constant shaking in water bath and switched to 5%CO2 every 10min. Papain/DNaseI was removed and samples were further diluted in 5ml EBSS2. Dissociated tissues were manually triturated (avoid air bubbles), followed by centrifugation at 900rpm for 10min at 4°C. Supernatant was discarded and the cell pellet gently resuspended in 200µl of EBSS2 until a smooth and creamy suspension was obtained. Dissociated cells were washed frequently with DMEM/F12 (Sigma) without phenol-red containing 3% fetal bovine serum (FBS; Life Technologies) and centrifuged at 900rpm for 10min at 4°C. Cell pellet was resuspended in 1ml of DMEM/F12 and filtered through a 70µm strainer cap (Corning) smoothly to harvest a clear cell suspension. Cell viability and yield was calculated by mixing an equal volume of acridine orange and propidium iodide and checked at the cell counter (Nexcelom Bioscience, MA, USA). Cell suspension (1ml) was diluted with 3ml PBS containing 0.04% BSA and centrifuged. Supernatant was removed and cell pellet resuspended in 400µl PBS containing 0.04% BSA. Cells were fixed by adding 1.6ml methanol dropwise and kept at -20°C for 30min and stored at -80°C for further use. The Methanol fixed cell suspensions were stored at -80°C for less than 7 days before processing. For each sample, approximately 1.5 million cells were taken for rehydration and downstream scRNA-seq experiment. Cells were first placed on ice and equilibrated to 4°C. After equilibration, cells were washed 2 times by 1ml pre-chilled rehydration buffer (1.0% BSA, 0.5U/µl RNAse inhibitor in 1x Dulbecco's Phosphate-Buffered Saline, DPBS). Centrifugation was done at 3000rcf for 10min at 4°C. Finally, cells were resuspended in pre-chilled 1xDPBS with 0.04% BSA and 0.5U/µl RNase inhibitor to achieve a concentration of 1,000 cells/µl. Rehydrated cells were immediately used for the scRNA-seq using the 10X genomics chromium single-cell gene expression platform. Around 35,000 cells from each sample were loaded on 1 well of 10X Single Cell A chip, where each single-cell was lysed and its transcriptome combined with a single barcoded gel bead in an oil droplet. Barcode cDNA libraries were then prepared using Chromium Single Cell 3' Library and Gel-beads kit v2 according to the manufacturer's instructions. Library quality was checked using Agilent High Sensitivity DNA chip on Agilent 2100 Bioanalyzer. High quality libraries were sequenced on Illumina HiSeq 4000 sequencer with an average depth of 200,000 reads per cell.