GSM7950112: ARC cDNA; Mus musculus; RNA-Seq
Male C57BL/6J mice aged 10-12 weeks were euthanized via transcardial perfusion using ice-cold HEPES-Sucrose Cutting Solution containing NaCl (110 mM), HEPES (10 mM), glucose (25 mM), sucrose (75 mM), MgCl2 (7.5 mM), and KCl (2.5 mM) at a PH of 7.4. Subsequently, brains were quickly dissected in the same solution, frozen using liquid nitrogen, and stored at -80 °C until nuclei isolation. To isolate nuclei, as previously described, the samples were thawed on ice, resuspended in HD buffer containing tricine KOH (10 mM), KCl (25 mM), MgCl2 (5 mM), sucrose (250 mM), 0.1% Triton X-100, SuperRNaseIn (0.5 U/ml), RNase Inhibitor (0.5 U/ml). Homogenization was performed using a 1-ml dounce homogenizer. The resulting homogenates were filtered using a 40 μM filter, centrifuged at 600g for 10 minutes, and resuspended in nucleus storage buffer containing sucrose (166.5 mM), MgCl2 (10 mM), Tris buffer (pH 8.0, 10 mM), SuperRNaseIn (0.05 U/ml), RNase Inhibitor (0.05 U/ml) for subsequent staining. Nucleus quality and number were assessed using an automated cell counter (Countess II, Thermo Fisher). For staining, nuclei were labeled with Hoechst 33342 (Thermo Scientific H3570; 0.5 µl per million nuclei), anti-NeuN Alexa Fluor 647-conjugated antibody (Abcam ab190565) (0.5 µl per million nuclei), and TotalSeq anti-Nuclear Pore Complex Proteins Hashtag antibody (BioLegend 682205) (0.5 mg per million nuclei) for 15 minutes at 4 °C. Following staining, samples were washed with 10 ml 2% BSA (in PBS) and centrifuged at 600g for 5 minutes. Nuclei were then resuspended in 2% BSA (in PBS) with RNase inhibitors (SuperRNaseIn 0.5 U/ml, RNase Inhibitor 0.5 U/ml) for subsequent fluorescence-activated cell sorting. The samples were gated based on Hoechst fluorescence to identify nuclei and then further sorted based on high Alexa Fluor 647 expression, designating NeuN+ nuclei as neurons. Nuclei were captured and barcoded using 10x Genomics Chromium v3 following the manufacturer's protocol. Libraries were prepared using 10x Genomics Chromium v3 following the manufacturer's protocol.