Fresh mouse brains were cryopreserved using OCT and sectioned into slides. Nuclei were then isolated following the manufacturer's protocol using the 10X Genomics Chromium Nuclei Isolation Kit with RNase Inhibitor (1000494). To prepare a cDNA library using the 10X Genomics Chromium Next GEM Single Cell 3' Kit v3.1 (1000269), the workflow typically involves the following key steps: Cell or Nuclei Preparation: Single cells or nuclei are partitioned into Gel Bead-In-Emulsions (GEMs) using the Chromium Controller. Each GEM contains unique molecular identifiers (UMIs) that label each cell's RNA individually. Reverse Transcription: Within the GEMs, reverse transcription occurs, converting RNA to cDNA while incorporating the UMIs and barcodes that tag each transcript to its respective cell. cDNA Amplification: After GEM disruption, cDNA is purified and amplified via PCR to generate sufficient material for library construction. Library Construction: Amplified cDNA is fragmented, end-repaired, and ligated with Illumina sequencing adapters. Dual indexing can be applied if needed, enabling multiplexing during sequencing. Quality Control: The final libraries are quantified using tools like the Agilent Bioanalyzer or TapeStation and prepared for sequencing on an Illumina platform.​