title: GSM5979843: ctrl-brain-1; Mus musculus; RNA-Seq
library_construction_protocol: Single cells were obtained according to a previous protocol with brief modification, Briefly, the mouse was deeply anesthetized by isoflurane and decapitated. After the brain was removed, the hippocampus was rapidly dissected under microscope. The tissue was cut into pieces on ice and then transferred to a 1.5 mL tube containing 2 mg/mL pronase (Sigma, Cat# P6911-1G), 50 μg/mL DNaseІ (Sigma, cat. no. D5025) in 1 mL HABG who consist of Hibernate A (Invitrogen, cat. no. A1247501) and B27 (Invitrogen, cat. no. 17504). The tube was then shaked for 30 min at 30℃ in a shaker at the speed of 200 rpm. After digestion, the hippocampus was gently triturated by Pasteur pipette tips to obtain the single cells. The cells were purified by OptiPrep (Sigma, cat. no. D1556) density gradient centrifugation at 800 g for 15 min, the single cells were resuspended in calcium and magnesium-free 1x PBS containing 1% BSA, and centrifuged for further purifying at 200 g for 2 min. The precipitated single cells were resuspended in 50 - 100 μL 1x PBS containing 1% BSA. Total and viability cell numbers were counted, and only samples with cell survival rates above 80% were used for subsequent sequencing. Total and viability single cell numbers were counted, and samples with cell survival rates above 80% were used for subsequent procedures. The qualified cells were washed and resuspended in PBS at a final concentration of 700~1200 cells/μL for 10x Genomics Chromium™ system operation. Then GEMs (Gel Beads-in-emulsion) were constructed for single cell isolation. Once the GEMs were successfully formed, they were collected and performed the reverse transcription to achieve labeling in a PCR machine. After that, the GEMs were damaged, then, magnetic beads were used to purify and enriched the first strand cDNA. Finally, they were subjected to cDNA amplification and quality control. A 10x Genomics Single Cell 3′ v2 Reagent Kit were applied for library construction by quality verified cDNAs, followed by fragmentation, adapters addition, and samples PCR indexing. Finally, the Illumina NovaSeq platform PE150 sequencing mode was performed for DNA sequencing and the sequencing volume criterion was set to > 50 k reads/cell.