Nanopore long-read RNA sequencing shows region-specific sex differences in wild-type mouse brain mRNA isoform expression and usage
To address differences in splicing across brain regions (cerebellum, cortex, hippocampus, and striatum) and sexes, we used long-read Oxford Nanopore Technologies (ONT) RNA sequencing to sequence 40 wild-type mouse brain cDNA libraries from 10 mice and calculated differential expression and transcript usage. We found that there is differential gene expression, differential transcript expression, and differential transcript usage across all brain regions. We found that the brain region with the most differential expression and transcript usage is the cerebellum, potentially driven by differences in cell type composition. Additionally, our findings suggest there is much differential splicing across brain regions and to a lesser extent, within brain regions across sexes. Overall design: We obtained flash-frozen hippocampus, striatum, cerebellum, and cortex wild-type C57BL/6J mouse brain tissues from The Jackson Laboratory (JAX #000664, age = 20 weeks) from five male and five female mice. We loaded 11 µl of each pooled library with 1 µl Rapid Adapter T (12 µl total) each onto 12 R9.4 flow cells (FLO-MIN106D). Because the Oxford Nanopore Technologies GRIDion (GRD-MK1) sequencing device can only hold five flow cells at a time, we sequenced these libraries in three separate sequencing runs (2, 5, and 5 flow cells at a time) for 72 hours each.