SnRNA seq of Patz1 injured Mice
Single-nucleus RNA sequencing of motor cortex begins with rapid tissue dissection followed by gentle homogenization in nuclear isolation buffer containing detergent and RNase inhibitors. Isolated nuclei are filtered through a series of cell strainers (40-30m) and purified via density gradient ultracentrifugation to minimize debris contamination. The purified nuclei suspension undergoes quality control assessment via fluorescence microscopy with DAPI staining. High-quality nuclei are then processed using droplet-based microfluidics (10x Genomics Chromium platform) for single-nucleus capture, barcoding, and cDNA synthesis. Following library preparation, sequencing is performed on an Illumina NovaSeq to generate approximately 50,000 reads per nucleus. Computational analysis includes quality filtering, dimensionality reduction, clustering, and cell type annotation based on marker gene expression to identify distinct neuronal and glial populations within the motor cortex.