Transcriptome Sequencing Protocol for Gene Expression

For each sample, 50 ng of total RNA was reverse transcribed using the Ion AmpliSeq Transcriptome Human Gene Expression kit following the protocol of the manufacturer (Thermofisher Scientific®). The cDNA libraries were amplified and barcoded using Ion AmpliSeq Transcriptome Human Gene Expression core panel and Ion Xpress Barcode Adapter (Thermofisher Scientific®). The amplicons were quantified using Agilent High Sensitivity DNA kit before the samples were pooled in sets of eight. Emulsion PCR and enrichment was performed on the Ion OT2 system instrument using the Ion PI Hi-Q OT2 200 kit (Thermofisher Scientific®). Samples were loaded on an Ion PI v3 Chip and sequenced on the Ion Proton System using Ion PI Hi-Q sequencing 200 kit chemistry (200 bp read length; Thermofisher Scientific®). The quality control of the sequencing data was evaluated as described previously (57 (link)). The DEG between conditions was calculated with (v1.18.1 using R v3.4.1). Genes were considered differentially expressed when their adjusted P-value was <0.05 and their|log2FoldChange|was >0.4 (options: lfcThreshold = 0.4, altHypothesis = ‘greaterAbs’). Data are available on GEO database (GSE161029).