GSM9228051: EnrichedNeuron_Esrra_shRNA_5; Mus musculus; RNA-Seq
After termination with carbon dioxide, each mouse was perfused with chilled PBS for 3 minutes. The brain tissue was harvested carefully and dissected into 8-10 sagittal slices with the help of a murine brain matrix (Ted Pella). Prefrontal cortex tissues where stereotaxic injection of Esrra shRNA was conducted of one mouse was polled in a petri dish filled with D-PBS (Dulbecco's Phosphate Buffered Saline with calcium and magnesium, supplemented with 1 g/L glucose and 36 mg/L sodium pyruvate). The dishes were placed on ice until further downstream processing. Tissue dissociation was performed with the Adult Brain Dissociation Kit for mouse (Miltenyi Biotec), following the manufacturer's instructions. Prefrontal cortex tissues of each mouse was transferred to a gentleMACS C Tube (Miltenyi Biotec) containing both enzyme mixes from the kit for enzymatic digestion. Mechanical enzymatic tissue dissociation was performed using program 37C_ABDK_01 of the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec). After dissociation, each brain tissue homogenate was applied to one 70 µm cell strainer (Corning, Corning, MA, USA). For detailed protocol, please see the methods section of the publication. Frozen whole cortex tissues from the test subjects of the AOF/VCD model, laser microdissection enriched GTP-positive cortex and hippocampal tissues from the AAV-study model or enriched neuronal populations were sent to the Novogene for total RNA extraction and RNA sequencing with the Illumina HiSeq X Ten platform. FASTQ files obtained were subsequently analysed by the FastQC (V. 0.11.9) of Babraham Bioinformatics, serving as a quality control assessment tool for these data. After assuring the quality of the data files, genome indexes were generated using the STAR software using. These indexes were generated with the mouse annotation and mouse genome assembly provided by the Gencode Release 28 (GRCm39, released in May 2021). The cleaned dataset was then aligned with the genome indexes using the STAR alignment software. FeatureCounts was then used to quantify the sequencing data. Data extraction, matrix construction and differential gene expression analysis was then performed using the DESeq2 package with standard procedures on R.