GSM8239680: 28.8 WT Sham; Mus musculus; RNA-Seq
Brain dissection and hippocampal cell dissociation were performed in 6 separate experiments. In each experiment we utilized the contralateral hemisphere as an internal control for each biological replicate. No randomization or blinding were performed during sample collection as no comparisons of different conditions were performed. For single-cell dissociation of the hippocampus, the mice were anesthetized with isoflurane, and the brain was quickly removed and transferred into ice-cold Hibernate A medium under a dissection microscope and then subjected to tissue dissociation. Hippocampal tissues were enzymatically digested using the Adult Brain Dissociation kit and mechanically disrupted with the gentleMCAS Dissociator according to the manufacturer's instructions. Briefly, tissue from each sample as loaded into a C tube with Enzyme 1 and 2. Tissue was subsequently dissociated on the gentleMACS Octo Dissociator with Heaters for 20-100 mg tissue. Following dissociation, samples were strained through a 70mm filter with dPBS and centrifuged at 300g x 10 min at 4oC. The supernatant was removed, and the tissue was resuspended in an appropriate amount of dPBS (<400mg, 1550 mL). Next, Debris Removal Solution was added (<400mg, 450mL) and overlaid with dPBS being careful not to disrupt the lower layer and centrifuged at 3000g x 10min at 4oC.  After removing the top two layers and adding fresh dPBS, the cells were spun at 1000g x 10 min and re-suspended in 0.2 ml of dPBS with 0.05% BSA. A 15-μl cell suspension was stained with trypan blue, and the live cells were counted. In all samples, cells were greater than 90% viable. During the entire procedure, the tissues and cells were kept in ice-cold solutions. The cell suspension was diluted with DPBS containing 0.05% BSA for single-cell capture. Cell suspensions were assessed using the Nexcelom Cellometer K2. The suspensions were prepped following 10X Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide.  Briefly, cells are partitioned into nanoliter gel beads-in-emulsion (GEMs). A pool of ~3,500,000 10x Genomics barcodes are used to index each individual cell separately and uniquely. Libraries are then created and sequenced on the Illumina Novaseq 6000, with 10x barcodes used to assign reads back to individual partitions, and therefore, to each individual cell. More than 10,000 single-cell events derived from hippocampal cells of mice were captured for recovery.