GSM8026393: hippocampus, Sublibrary 3; Mus musculus; RNA-Seq
Hippocampi (n=3) were extracted and pooled. Pooled samples were homogenized using dounce homogenizers and a homogenization buffer (1.5 M NIM1 Buffer with 250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris Buffer pH 8, 1uM DTT, 0.4U/uL Enzymatics RNAase-Inhibitor (#Y9240L), 0.2U/uL Superase-Inhibitor (Thermo Fisher Scientific, #AM2694), and .1% Triton X-100). Hippocampi homogenates were filtered (40uM strainer) and clarified. Resulting homogenates were resuspended in a PBS buffer with RNase Inhibitors (0.05U/ul of Enzymatics RNAase-Inhibitor and Superase-Inhibitor) and re-pelleted. To remove myelin debris, homogenates were incubated with Myelin Removal Beads II (Miltenyi Biotec, #130-096-731) for 15 minutes at 4°C. Homogenates were washed (50% PBS and 50% PBS + 1% BSA) and re-pelleted. Supernatant was removed and samples were resuspended in wash buffer. One LS column (Miltenyi Biotec, # 130-042-401) per sample was used to filter the samples followed by pelleting and resuspending in wash buffer. Nuclei were counted with AO/PI (Logos Biosystems, #F23001) on a Luna-FL Cell Counter and fixed with a Nuclei Fixation Kit (Parse Biosciences, #SB1003) per the manufacturer's instructions followed by freezing at -80°C in a Mr. Frosty (Thermo Fisher Scientific, #5100-001). Parse Biosciences Whole Transcription Kit was used to barcode and generate sub-libraries with 12,500 nuclei/sub-library comprised of all samples. snRNAseq