4.6. RNA-Seq Analysis

For the preparation of the library, we used the TruSeq RNA Exome protocol (Illumina, San Diego, CA, USA) and the Illumina MiSeq instrument for library sequentiation. We took advantage of the fastQC software (version 0.11.5, Babraham Institute, Cambridge, UK.) to confirm the parameters of the raw data in Fastq format. With Trimmomatic (version 0.38, Usadel Lab, Aachen, Germany) [67], we removed the adapters and the base with low-quality scores. Then, we aligned the reads with spliced transcripts alignment to a reference (STAR)RNA-seq aligner (version 2.7.3a, New York, NY, USA) [68] choosing the human reference genome version GRCh38, and we performed the transcripts count with the python package htseq-count (version 0.6.1p1, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany) [69]. The analysis of the differentially expressed genes (DEGs) was computed through the package DESeq2 of Bioconductor [70] in R (version 3.6.3, R Core Team). We used the post-hoc Benjamini–Hochberg method, and we kept as significant only the DEGs whose q-value was not higher than 0.05. We did not use any fold change cut-off.