Transcriptome analysis by AmpliSeq

Transcriptomic analysis of HUVEC cells treated with or without COCO for 16 h, in quadruplicate, was performed using the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific) according to manufacturer's instructions. Briefly, mRNA from 10 ng of total RNA was reverse transcribed using SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) and amplified with Ion AmpliSeq HIFI Mix together with primers from the Ion AmpliSeq Transcriptome Human Gene Expression Core Panel simultaneously targeting over 20,000 RefSeq genes. Primer sequences were partially digested with FuPa Reagent and then barcoded using Ion Xpress Barcodes (Thermo Fisher Scientific). Purification was carried out by AMPure XP Reagent (Beckman Coulter). Libraries concentrations were defined by qPCR using Ion Library Quantification kit (Thermo Fisher Scientific). Libraries were pooled together for emulsion PCR, carried out using the Ion Chef Instrument (Thermo Fisher Scientific). Purified Ion Sphere Particles were loaded on Ion P1 Chip. The sequencing was performed on Ion Proton system (Thermo Fisher Scientific). Ion Torrent software, Torrent Suite v5.12 (Thermo Fisher Scientific), was used for base calling, alignment to the human reference genome (hg19) and quality control. Raw reads were then analyzed automatically using the AmpliSeqRNA plugin to generate gene‐level expression values. Differential gene expression was determined using DEseq2 (version 3.11) package. Gene set enrichment analysis (GSEA) was performed using the GSEA software using pre‐defined gene sets based on prior biological knowledge (version 4.1).