title: GSM7864620: WT_24W_1; Mus musculus; RNA-Seq
library_construction_protocol: Nuclei were isolated from frozen tissue as previously described.10 For mouse snRNA-seq, hemicerebella of three animals per genotype (WT and SCA1 KI) per timepoint (5, 12, 18, 24, and 30 weeks) were used, for a total of 30 animals. For human post-mortem samples, pieces of cerebellar cortex were harvested from frozen tissue of ten unique individuals per diagnosis (CTRL and SCA1), for a total of 20 human samples. Briefly, frozen mouse or human tissue was gently dounce homogenized in 2mL of ice-cold Nuclei EZ Prep buffer (Sigma; NUC101-1KT) with large clearance pestle “A” and then a small clearance pestle “B” for 25 strokes each, then incubated on ice for five minutes with an additional 2mL of cold EZ Prep buffer. Nuclei were centrifuged at 500xg for five minutes at 4°C. Pelleted nuclei were then washed in 4mL of cold EZ Prep buffer, incubated on ice at five minutes, and centrifuged again at 500xg for five minutes at 4°C. Pelleted nuclei were then washed in 4mL of Nuclei Suspension Buffer (NSB) containing 1x phosphate buffered saline (PBS), 0.01% BSA, and 0.1% RNase inhibitor (Clontech/Takara; 2313B) and centrifuged at 500xg for five minutes at 4°C. Finally, nuclei were resuspended in 1mL of NSB, filtered through a 40mm cell strainer (Fisher Scientific; 22-363-547), and counted using a Countess II FL Cell Counter (Applied Biosystems; A27978). Single-nucleus suspensions were diluted to approximately 1000 nuclei per ml in NSB. Libraries were prepared from diluted single-nucleus suspensions using the 10x Genomics Chromium Single Cell 3' Reagent Kits v.3 (mouse dataset) or v.3.1 (human dataset) through the Yale Center for Genome Analysis (YCGA). Briefly, 10,000 cells per sample were added to RT Master Mix, loaded on the Single Cell A Chip, and portioned with a pool of about 750,000 barcoded gel beads to form nanoliter-scale Gel Beads-In-Emulsions (GEMs). Each gel bead has primers containing the following: an Illumina R1 sequence (read 1 sequencing primer), a 16-nucleotide barcode, a 12-nucleotide unique molecular identifier (UMI), and a 30-nucleotide poly-dT primer sequence. Upon dissolution of the Gel Beads in a GEM, the primers were released and mixed with the cell lysate and Master Mix. Incubation of the GEMs then produced barcoded, full-length cDNA from poly-adenylated mRNA. Silane magnetic beads were then used to remove leftover biochemical reagents from the post-GEM reaction mixture. Full-length, barcoded cDNA was then amplified by PCR to generate sufficient mass for library construction, and enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size prior to library construction. P5, P7, a sample index, and R2 (read 2 primer sequence) were added during library construction via end repair, A-tailing, adaptor ligation, and PCR. The final libraries contained the P5 and P7 primers used in Illumina bridge amplification. Libraries were sequenced using the NovaSeq6000 at the YCGA. Sample concentrations were normalized to 1.2nM and loaded onto an Illumina NovaSeq S4 flow cell at a concentration that yielded 20k-50k passing filter clusters per cell per sample. Samples were sequenced using paired-end sequencing on an Illumina NovaSeq6000 instrument according to Illumina protocols using 10x sequencing specifications. The 8bp index was read during an additional sequencing read that automatically followed the completion of read 1. Data generated during sequencing runs were simultaneously transferred to the YCGA high-performance computing cluster. A positive control (prepared bacteriophage Phi X library) provided by Illumina was spiked into every lane at a concentration of 0.3% to monitor quality in real time. Signal intensities were converted to individual base calls during a run using the system's Real Time Analysis (RTA) software. Base calls were transferred from the machine's dedicated personal computer to the Yale high-performance computing cluster via a 1 Gigabit network mount for downstream analysis. snRNA-seq