RNA-Seq Protocol for Differential Expression

Between 3 and 5 μg of total RNA were sent to Admera Health LLC (NJ, USA) for sequencing in paired-end mode, 2x 150 nucleotides, using an Illumina HiSeq 2500 sequencer. The sequencing library was prepared with the NEBNext Ultra II kit, with RiboZero rRNA depletion (NEB #E7103, Illumina #20040526). Between 60 and 90 million paired end reads were obtained from each sample and mapped to the human genome (Hg38) with STAR v2.7.5a (98 (link)) using default parameters. Mapping quality was assessed with FastQC v0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc). Sequencing adaptors were removed with trimmomatic v0.35 (99 (link)). Low-quality reads and mitochondrial contaminants were removed, leaving on average 70 million useful reads per sample (Supplementary Material, Table S1). Differential expression analysis was performed in R with DESeq2 v1.32.0 (100 (link)) after transcript quantitation with Salmon v1.4.0 (101 (link)). We used an FDR threshold of 5% for differential expression.