Peripheral blood gene expression profiling predicts response to mycophenolate in systemic sclerosis related interstitial lung disease

PBC RNA samples from 134 baseline and 98 month-12 visits, corresponding to the active treatment period of both arms in Scleroderma Lung Study II, along with 45 healthy controls were investigated by global RNA sequencing. The objective was to examine the peripheral blood cell (PBC) gene expression changes ensuing from mycophenolate mofetil (MMF) or cyclophosphamide (CYC) treatment and to determine the predictive significance of baseline PBC transcript scores for response to immunosuppression in systemic sclerosis (SSc) related interstitial lung disease (ILD). Overall design: Whole blood samples were collected in PAXgene tubes. PBC RNA was extracted according to the manufacturer's protocol. RNA integrity was measured on an Agilent 2100 Bioanalyzer (Agilent Technologies). mRNA was enriched from total RNA using oligo(dt) beads (NEBNext Ultra II RNA Kit following the poly(A) enrichment workflow). The mRNA was subsequently fragmented randomly in fragmentation buffer, and reverse transcribed to cDNA. The cDNAs were converted to double stranded cDNAs, then subjected to end-repair, A-tailing, and adapter ligation, size selection and PCR enrichment. Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies), and then diluted to 1 ng/ul before checking insert size on an Agilent 2100 and quantifying to greater accuracy by quantitative PCR (q-PCR) (library activity >2 nM). Libraries are pooled into Novaseq6000 machines according to molarity and expected data volume. A paired-end 150 bp sequencing strategy was used to generate an average of 99 million reads per sample.