RNA sequencing

Total RNA was isolated from MEPM cells 24 h after mimic (n = 2) or scramble transfection (n = 2). RNA sequencing libraries were prepared using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (KAPA Biosystems) according to protocol. Fragmentation and priming were performed at 94°C for 6.5 min. First-strand synthesis, second-strand synthesis and A-tailing were performed according to protocol. For the adapter ligation, a 7 μM stock was used (NextFlex DNA barcodes, Bioo Scientific). First, and second post-ligation cleanup was performed according to protocol. For the library amplification, six cycles were used. The library amplification cleanup was performed using a 0.8× bead-based cleanup. Library size was determined using the High Sensitivity DNA bioanalyzer (Agilent Technologies), library concentration was measured using the DeNovix dsDNA High Sensitivity Assay (DeNovix). Paired-end sequencing was performed using an Illumina NextSeq 500. Low-quality filtering and adapter trimming were performed using Trim Galore! v0.4.5 (Babraham Bioinformatics), a wrapper tool around the tools Cutadapt v1.18 and FastQC v0.11.8 (Babraham Bioinformatics). Reads were mapped to a human reference genome (GRCh38.95, Ensembl) with Star v2.7.5a, resulting in BAM files. BAM files were counted with HTSeq [HTSeq-count tool v0.11.0] with default parameters using a complementary gtf file, containing annotation for GRCh38.95 (Ensembl). Counts were normalized using gene length and transcripts per million were produced. MultiQC (quality control) was used to combine results and quality checks of all the samples.