Sequencing libraries were prepared with TruSeq Stranded Total RNA kit (Illumina) using 200ng total RNA. Qualities of sequencing libraries were assessed with 4200 TapeStation with the DNA1000 reagent kit. RNA processing was carried out using Illumina NextSeq 500 Sequencing. FastQ files were generated via Illumina bcl2fastq2 (Version 2.17.1.14 - https://support.illumina.com/downloads/bcl2fastq-conversion-software-v2-20.html) starting from raw sequencing reads produced by Illumina NextSeq sequencer. Qualities of individual sequences were evaluated using FastQC software (see Code Availability 1) after adapter trimming with cutadapt software. Gene and transcript intensities were computed using STAR/RSEM software 45 using Gencode Release 27 (GRCh38) as a reference, using the “-strandness forward” option. Differential expression analysis for mRNA was performed using R package DESeq2 46, selected because of its superior performance in identifying isoforms differential expression 47. Genes were considered differentially expressed and retained for further analysis with |log2(nichoid sample/control sample)| ≥ 1 and a FDR ≤ 0.1. We imposed minimum |Log2FC| of 1 and a FDR lower than 0.1 as thresholds to differentially expressed genes. This choice is motivated by the decision to maximize the sensitivity of this analysis, in order to perform a massive screening and identify candidate genes to be validated with a wider sample population with real-time analysis. The raw data obtained from the RNA-seq analysis is deposed on the Gene Expression Omnibus repository (GSE150767).

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