title: HD_1_B_S12
design: Mouse motor cortex and striatum tissue samples were homogenized on ice in sucrose-based buffer containing Triton X-100 and RNase inhibitors. Tissue was processed using dounce homogenizers with loose and tight pestles, then filtered through 40 uM strainers. Nuclei were pelleted by centrifugation and washed three times in PBS with BSA and RNase inhibitors. For long-term storage, nuclei were fixed in formaldehyde, permeabilized with Triton X-100 and resuspended in storage buffer with DMSO. Samples were stored for up to six months.Split-pool ligation-based barcoding was performed in multi-well plates following an established protocol (Rosenberg et al.) with optimizations. Round 1 added well-specific barcodes through reverse transcription using barcoded poly(dT) primers, with 5-6K nuclei loaded per well across multiple wells. The plate was incubated in a thermocycler, then all reactions were pooled. Rounds 2 and 3 added DNA barcodes via ligase reactions, with Round 3 incorporating unique molecular identifiers and biotin tags for purification. After three barcoding rounds, nuclei were redistributed into sublibraries and lysed with Proteinase K and SDS.Biotin-tagged cDNA was purified using streptavidin beads and underwent template-switching. PCR amplification used high-fidelity polymerase with initial cycles followed by additional optimization. Libraries were fragmented, underwent end-repair and A-tailing, and received Illumina adapters through ligation. Following indexed PCR, bead cleanup selected specific fragment size ranges.Sequencing used paired-end reads targeting thousands of reads per nucleus. Read 1 captured transcript sequences while Read 2 contained barcodes and UMIs for demultiplexing. A custom computational pipeline processed the data with error-correction allowing single base mismatches and applying minimum read thresholds per barcode. The pipeline performed barcode extraction, sequence mapping, feature assignment, and UMI collapse to generate gene expression count matrices for downstream single-cell analysis.