GSM7102022: Acute DR;  Hippocampus Sample 2 24 mo; Mus musculus; RNA-Seq
Single-nuclei preparation and sequencing was performed as previously described (Hahn et al., 2020) with the following modifications: Nuclei from whole hippocampus were isolated with EZ Prep lysis buffer (Sigma, NUC-101) on ice. Samples were placed into 2 ml cold EZ lysis buffer in a 2 ml glass dounce tissue grinder (Sigma, D8938) and homogenized by hand 25 times with pestle A followed by 25 times with pestle B while incorporating a 180-degree twist. Tissue homogenate was transferred to a fresh 15 ml tube on ice. The tissue grinder was rinsed with 2 ml fresh lysis buffer and transferred to the tube holding the homogenate for a total volume of 4 ml. Samples were incubated on ice for 5 minutes. Nuclei were centrifuged at 500 x g for 5 minutes at 4°C, supernatant removed and pellet resuspended with 4 ml EZ lysis buffer, and incubated on ice for 5 minutes. Centrifugation at 500 x g for 5 minutes at 4°C was repeated. After removing supernatant, the pellet was resuspended with 4 ml chilled PBS and filtered through a 35-um cell strainer into a 5 ml round bottom FACS tube (Corning, 352235). Following centrifugation at 300 x g for 10 minutes at 4°C with break 3, supernatant was gently poured out leaving behind the nuclei pellet. Pellet was resuspended in 400 ul PBS containing 1% BSA (Thermo Fisher, BP9700100), 0.2 ul Hoechst dye (Thermo Fisher, H3570) , and 2 ul recombinant RNase inhibitor (Takara, 2313B). Isolated nuclei were sorted on a MA900 Multi-Application Cell Sorter (Sony Biotechnology). 50k single nuclei per sample were collected into 1.5 ml DNA lo-bind tubes (Eppendorf, 022431021) containing 1 ml buffer mix with PBS, UltraPure BSA (Thermo Fisher, AM2618), and RNase inhibitor (Takara, 2313B). Collected nuclei were centrifuged at 400 x g for 5 minutes at 4°C with break 2. Supernatant was removed leaving 40 ul suspended nuclei. Nuclei were counted using a hemocytometer (Sigma, Z359629-1EA) and assessed for concentration and quality. Reagents of the Chromium Single Cell 3' GEM & Gel Bead Kit v3.0 (10X Genomics,) were thawed and prepared according to the manufacturer's protocol. Nuclei and master mix solution was adjusted to target 10,000 nuclei per sample and loaded on a standard Chromium Controller (10X Genomics, 1000204) according to manufacturer protocols. We applied 11 PCR cycles to generate cDNA. Library construction was conducted using Chromium Single Cell 3' Library Construction Kit v3 (10X Genomics, 1000121). All reaction and quality control steps were carried out according to the manufacturer's protocol and with recommended reagents, consumables, and instruments. We chose 11 PCR cycles for library generation. Quality control of cDNA and libraries was conducted using a Bioanalyzer (Agilent) at the Stanford Protein and Nucleic Acid Facility. 10X Nuc-seq, v3.0 chemistry