GSM7034363: N_50; Homo sapiens; RNA-Seq
PreSSc's and healthy controls' whole blood samples were col_x0002_lected in PAX gene tubes and stored at –80_x0002_C. RNA was extracted according to the manufacturer's protocol and quality was assessed by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Globin genes were depleted using GLOBIN Clear kit. mRNA was enriched from total RNA using oligo(dt) beads (NEB Next 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, Carlsbad, CA, USA), and then diluted to 1 ng/ul before checking insert size on an Agilent 2100 and quantified to greater accuracy by quantitative PCR (q-PCR) (library activity >2nM). Libraries were 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 88 million reads per sample.