GSM8986744: GroupB, mouse 4, RNA, replicate 2; Mus musculus; RNA-Seq
Adult mice were euthanized by cervical dislocation, and bilateral samples of the frontal cortex (spanning bregma 1.90 to 1.40) were rapidly dissected and flash-frozen at –80°C for later processing. For transcriptomic analysis via RNA-seq, frozen frontal cortex tissue was processed to isolate neuronal nuclei. Fluorescence-activated cell sorting (FACS) was used to enrich for NeuN-positive (NeuN⁺) neuronal populations. Each RNA-seq replicate was prepared from approximately 6,000 sorted neuronal nuclei. For transcriptomic analysis via RNA-seq, frozen frontal cortex tissue (bregma 1.90–1.40) was processed to isolate neuronal nuclei. Fluorescence-activated cell sorting (FACS) was used to enrich for NeuN-positive (NeuN⁺) populations. A total of 100 μL of sorted neuronal nuclei suspension (~12,000 nuclei) was used for RNA extraction using the RNeasy Mini Kit (Qiagen) and RNase-Free DNase Set, following the manufacturer's instructions. RNA from ~6,000 nuclei (in 30 μL) was concentrated by ethanol precipitation and resuspended in 4.6 μL of RNase-free water. Libraries were prepared using the SMART-seq v4 Ultra Low Input RNA kit (Clontech) and the Nextera XT DNA Library Preparation kit (Illumina), with ~600 pg of purified cDNA used per replicate. Library quality was assessed using the Agilent High Sensitivity DNA kit on a TapeStation system. Libraries were pooled with ChIP-seq samples (18–22 total per run), quantified with a KAPA Library Quantification Kit, and sequenced on an Illumina HiSeq 4000 (single-end, 50 bp), yielding ~11 million reads per RNA-seq sample.