GSM8794717: Conventionally raised, rep 2; Mus musculus; RNA-Seq
Brains were perfused with PBS then the hippocampus was dissected and flash frozen in liquid nitrogen. Nuclei were isolated using an extraction protocol outlined in PMID: 28846090. Frozen tissue was placed in homogenization buffer (consisting of 0.26M sucrose, 0.03M KCl, 0.01M MgCl2, 0.02M Tricine-KOH pH 7.8, 0.001M DTT, 0.5mM Spermidine, 0.05mM Spermine, 0.3% NP40, and protease inhibitor) and homogenized using both pestle A (0.0030-0.0050 in. clearance) and pestle B (0.0005-0.0025 in. clearance) of a KIMBLE dounce tissue grinder (Sigma D8938). Density gradient centrifugation was performed using iodixanol and the nuclei band was captured at the 30%-40% iodixanol interface. Nuclei were washed in RSB buffer (0.01M Tris-HCL pH 7.5, 0.01M NaCl, 0.003M MgCl2, and 0.1% Tween-20) then samples were combined such that each treatment group was represented by two samples, each containing roughly equal numbers of cells from two mice within a single treatment group. After nuclei isolation, samples immediately underwent cell capture using the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics # PN-1000268) according to the manufacturer's guidelines. Approximately 1,600 cells were loaded onto a Chromium Next GEM Chip and run using a Chromium Controller and library preparation steps were performed according to the manufacturer's guidelines.