RNA-Seq Protocol for Whole Transcriptome Analysis

RNA (5 ng) was subjected to whole transcriptome amplification using NuGEN's Ovation RNA-Seq V2 kit (San Carlos, CA, USA) according to manufacturer's instructions. Briefly, cDNA was amplified from total RNA using a single primer isothermal amplification (SPIA). The amplified cDNA samples were subsequently purified using a MinElute Reaction Cleanup Kit (Qiagen; Valencia, CA, USA). The cDNA samples were fragmented into smaller pieces, blunt-ended, and ligated to indexed (barcoded) adaptors and amplified using an Ultralow System V2 kit according to the manufacturer's protocol. Final library size distribution was determined using an Agilent Bioanalyzer 2100. Five libraries from five different donors were sequenced on the Illumina NextSeq500 platform (San Diego, CA, USA). The FASTQ files were uploaded to UPPMAX and quality checked using fastQC (39 ). Trimmomatic (40 (link)) was used to remove adaptors and low-quality bases and the reads were then mapped to human reference genome hg19 using STAR (41 (link)). Counts for each gene were calculated using featureCounts (42 (link)). The data was normalized and differentially expressed genes determined using R/DeSeq2 (43 (link)). Analysis of pathways was done by Ingenuity Pathway Analysis (Qiagen), R analysis, Gene Ontology (GO) Enrichment Analysis (Geneontology.org), and custom gene lists.