title: GSM8713718: hippocampus, AAV-STOP; Mus musculus; RNA-Seq
library_construction_protocol: The mouse hippocampus was dissected and snap-frozen at -80°C. Tissue (100 mg) was powdered on dry ice using a scalpel and transferred to a 1.5-mL microtube. Homogenization was performed by adding 0.5 mL of freshly prepared Homogenization Buffer (320 mM Sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris pH 7.8, 0.1 mM EDTA, 0.1% NP40, 0.1 mM Protease inhibitor, 1 mM Beta-mercaptoethanol, 1 U/μL RNase inhibitor), followed by gentle pipetting and incubation on ice for 10 minutes. The homogenate was passed through a 30-μm strainer and centrifuged at 300g for 3 minutes at 4°C. The pellet was resuspended in 0.5 mL Homogenization Buffer and mixed with 0.5 mL Upper Gradient Centrifugation Buffer (50% OptiPrep Gradient, 5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris pH 7.8, 0.1 mM Protease inhibitor, 1 mM Beta-mercaptoethanol, 1 U/μL RNase inhibitor). The mixture was gently layered on top of 1 mL of Lower Gradient Centrifugation Buffer (29% OptiPrep Gradient, 1 U/μL RNase inhibitor) in a 2-mL microtube. The gradient was centrifuged at 7,200g for 15 minutes at 4°C. The supernatant was carefully discarded. The nuclei pellet was resuspended in the PBS containing 0.04% BSA and 1 U/μL RNase inhibitor to achieve a concentration of 700-1200 nuclei/μL. Nuclei (∼20,000 per sample) were loaded onto a 10x Genomics Next GEM chip G. Libraries were prepared using the Chromium Next GEM Single Cell 3ʹ Library and Gel Bead kit v3.1 (10x Genomics), following the manufacturer's protocol, and sequenced on an Illumina NovaSeq 6000.