Transcriptomic Insights into Membrane Transport Dysfunction in Spinocerebellar Ataxia Type 3 - Annexes and References

Author

Adriana A. Vaz

Published

June 26, 2026

ANNEX I

Experimental and bioinformatic workflow

Samples were obtained from 4 wild type and 4 transgenic SCA3 (hATXN3+/-) animals (10). Total RNA was extracted from cerebellar tissue, and its integrity was assessed using the Agilent TapeStation system (Agilent Technologies, USA). Library preparation was performed using the TruSeq Stranded mRNA kit (Illumina, USA), which employs poly-A enrichment to select for messenger RNA. Sequencing was carried out as paired-end 2 × 151 bp reads on the NovaSeq X platform (Illumina, USA). The resulting FASTQ files were processed using the nf-core/rnaseq pipeline (version 3.21.0) (16). Quality control of raw sequencing reads was performed with FastQC (version 0.12.1) (17), and adapter sequences and low-quality bases were trimmed using TrimGalore (version 0.6.10)(18). Trimmed reads were aligned to the mouse reference genome (GRCm39/mm39)(19) using STAR (version 2.7.11b)(20), and transcript-level expression was quantified with Salmon (version 1.10.3)(21), producing a raw count matrix and normalised expression values in transcripts per million (TPM). The raw quantification outputs from the nf-core/rnaseq pipeline were imported into RStudio (version 4.5.3) (15) for further analysis. Differential expression and functional enrichment analysis were performed with several R packages, such as tidyverse (version 2.0.0)(22), DESeq2 (version 1.50.2)(11), ComplexHeatmap (version 2.26.1)(23), clusterProfiler (version 4.18.4)(12), enrichplot (version 1.30.5)(13), and the org.Mm.eg.db (version 3.22.0)(14) database.

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