GSM9142693: M-A1-11SN; Mus musculus; RNA-Seq
The medial prefrontal cortex (mPFC) was microdissected from 500-µm coronal brain sections prepared using adult rodent brain matrices (R&D, 68717). Slices were transferred to ice-cold HBSS, and mPFC-containing regions were identified microscopically and isolated using a 23-gauge needle. For sample preparation, we established two biological replicates per experimental group, with each replicate consisting one male-derived sample (pooled mPFC tissue from 4 male mice) and one female-derived sample (pooled mPFC tissue from 4 female mice). Tissue from each animal was pooled, briefly centrifuged to remove HBSS, and flash-frozen in liquid nitrogen. Nuclei were isolated from frozen mPFC tissue using the Chromium Nuclei Isolation Kit (PN-1000494, 10x Genomics) strictly following the manufacturer's protocol. Tissue samples were homogenized in Lysis Buffer using a pestle and filtered through a column. Debris was removed by centrifugation in Debris Removal Buffer (300g, 10 min). Isolated nuclei were washed, resuspended in Nuclei Buffer, and counted. Nuclei suspensions were loaded directly onto the 10x Genomics Chromium platform. Single-nucleus RNA sequencing (snRNA-seq) libraries were prepared targeting a capture of approximately 10,000 nuclei. Single-cell gel beads-in-emulsion (GEMs) were generated using the Chromium Single Cell 3' Library & Gel Bead Kit v3.1 (10x Genomics, PN-1000268). Captured nuclei were lysed within GEMs, and released RNA underwent reverse transcription using primers containing a poly-dT sequence, a cell barcode, a unique molecular identifier (UMI), and the Read 1 primer sequence. Barcoded cDNA was recovered, purified, and amplified by PCR. Adapter ligation was then performed to add the sample index and Read 2 primer sequence. Following library quality control assessment, paired-end sequencing (150 bp reads) was performed on an Illumina NovaSeq 6000 platform (Berry Genomics Corporation, Beijing, China).