Rna seq

The RNA‐Seq was performed at Takara Bio Inc. (Shiga, Japan) or Macrogen, Inc. (Seoul, South Korea) by using HiSeq2000 or HiSeq4000 (Illumina Inc., San Diego, CA, USA). Splicing bioinformatics analysis using MISO and pathway analysis using Cytoscape (Shannon et al, 2003) were performed as previously described (Funnell et al, 2017). Briefly, the BAM alignment files produced by TopHat and the files for AS events (for hg19) provided by MISO were used as inputs. To identify significantly changed AS events between two samples, we used the following criteria: (i) the absolute value of the difference for PSI value (ΔPSI) between two samples was ≥ 0.1; (ii) the sum of inclusion and exclusion reads was > 10, with both inclusion and exclusion reads ≥ 1; and (iii) the Bayes factor (BF), which quantifies the odds of differential regulation occurring, was > 20. For clustering, we used 1,320 splicing events with ΔPSI > 0.4, the BF > 20, and SD of PSI for each splicing event > 0.2. Hierarchical clustering was performed using heatmap3 from the R package.

RNA-seq was performed at TaKaRa Bio. Inc. (Shiga, Japan). Read mapping on a genomic sequence was performed with DRAGEN Bio-IT software ver3.6.3 (Illumina, San Diego, CA). The count data were analyzed using the tag count comparison (TCC) R package^{35 (link)} through TCC-Graphical User Interface^{36 (link)}. The basic algorithm of TCC was previously described^{37 (link)}.