FASTQ files for pTRE, WT, KK, and R109K samples in three biological replicates were trimmed to remove adapters and low-quality bases with TrimGalore v.0.4.3 ( The trimmed reads were aligned to the human reference genome (build hg19) with STAR aligner v.2.5.1 (32) with two mismatches allowed. Differential gene expression analysis was performed with DESeq2 R Bioconductor package v.1.16.1 (33) using read counts data provided by the aligner. Genes were considered differentially expressed if the adjusted P value, calculated using the Benjamini-Hochberg method to minimize the false discovery rate (FDR), was less than 0.01, and the change in expression level was greater than twofold. Differential splicing analysis, Ψ calculation, and splicing events statistics were performed with rMATS turbo package v4.0.1 (17). The FDR threshold for differential percent spliced in PSI was chosen to be 0.01. The GO enrichment analysis was performed with MetaCore software suite (Clarivate Analytics, v.6.33-69110) to reveal biological processes overrepresented in differentially spliced gene sets. P values for GO enrichment analysis were calculated using the formula for hypergeometric distribution, reflecting the probability for a GO term to arise by chance. Statistically enriched terms were identified using a threshold FDR of 3%. Clustering of GO:BP terms was performed using the R Bioconductor goseq package (v.1.30), and annotations were provided in (v.3.5.0) and GO.db (v.3.5) packages. Gene expression data have been deposited in the National Center for Biotechnology Information’s (NCBI) Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE111961.

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