GSM9753763: RNA-seq prefrontal cortex, MOR-SAL, female, replicate 15-4; Mus musculus; RNA-Seq
Brain tissues (PFC, hippocampus, hypothalamus, striatum) were collected by bilateral punch (2.0 mm) and flash-frozen at -80C. Nuclei were isolated in cold extraction buffer using a Dounce tissue grinder, filtered (40 um), and enriched by iodixanol gradient centrifugation. NeuN+ neuronal nuclei were labeled with anti-NeuN Alexa Fluor 488 (MAB377X, EMD Millipore) at 4C for 1 h, then sorted by FACS (BD Biosciences). Nuclear RNA was extracted from ~5,000 NeuN+ nuclei per sample using RNeasy Mini Kit (74104, Qiagen) with on-column DNase digestion (RNase-Free DNase Set 79254, Qiagen). RNA-seq libraries were prepared from ~5,000 NeuN+ sorted nuclei using a modified Smart-seq3 protocol with tagmentation-based library construction. Reverse transcription was performed on nuclear RNA, followed by tagmentation with custom P5/P7-adapter-loaded Tn5 transposase (Diagenode) and PCR amplification with KAPA HiFi ReadyMix and barcoded primers. Libraries were size-checked on a TapeStation (G2992AA, Agilent) using the High Sensitivity DNA Analysis Kit (5067-4626), primer-dimers removed with SPRIselect beads, quantified with KAPA Library Quantification Kit (4824, Roche), pooled, and sequenced on an Illumina NovaSeq X Plus platform (paired-end 150 bp, ~15 million reads per library).