GSM9321744: TFE4 WT PU1; Mus musculus; RNA-Seq
For single nucleus RNA-sequencing (snRNA-seq), mouse brains were collected dissected to isolate the hippocampal regions. The brain slices were mechanically minced with surgical scissors in cold lysis buffer containing 10 mM Tris buffer (pH 7.4), 5 mM MgCl2, 25 mM KCl, 1 mM DTT, 5X EDTA-free protease inhibitor (Millipore Sigma, Cat# 11873580001), 0.02 U/µL RNase-In RNase inhibitor (Promega, Cat# N2518), and 0.02 U/µL SUPERase-In RNase Inhibitor (ThermoFisher Scientific, Cat# AM2696) in nuclease-free water. The suspension was then homogenized on ice using a glass dounce homogenizer (15 loose and 15 tight strokes). The homogenate was filtered through a pre-wet 40-µm cell strainer (Fisher Scientific, Cat# MT4040) and rinsed with cold wash buffer containing PBS, 0.1% BSA, RNase-In, and SUPERase-In. Nuclei were purified with Anti-Nucleus MicroBeads (Miltenyi Biotec, Cat# 130-132-997) per the manufacturer's protocol. The samples were further sorted with AF647-anti-NeuN (to isolate neuronal nuclei; Cat# ab190565) AT 1:1000 (v:v) and PE-anti-PU.1 (to isolate microglia nuclei; Cat# 57800S) at 1:50 (v:v). After sorting, nuclei quality and yield were confirmed using a Cellometer K2 fluorescent cell counter with AO/PI staining, and libraries were immediately prepared using Chromium GEM-X Single Cell 3' Kit v4 (10x Genomics) according to manufacturer's instructions. All snRNA-seq libraries were sequenced using Illumina NovaSeq X targeting 50,000 reads/nuclei through GTAC @ MGI at Washington University in St. Louis.