RNA extraction, sequencing and analysis
PBMCs were isolated from the blood of RA patients and healthy donors via
centrifugation over Ficoll-Paque Plus (GE Healthcare, Sweden). RNA was
extracted from PBMCs using Trizol: chloroform, precipitated in
isopropanol. Sample quality was assessed using Nanodrop. Total RNA was
enriched for mRNA using poly-A selection. In brief, mRNA was fragmented,
reverse transcribed, adapted with sequencing primers and sample
barcodes, size selected, and PCR enriched. Libraries were sequenced on
the HiSeq 2000 platform. RNA sequence reads were aligned to the human
reference genome (NCBI, hg19) using TopHat2 aligners. Normalised gene
counts were calculated using HTSeq, and differential gene expression
between samples was identified via edgeR. Gene expression (RPKM) values
were calculated using Cufflinks v2.0.2. Gene Ontology
(http://geneontology.org/) associations were determined using Gorilla.
Network construction was based on the Ingenuity Pathway Analysis
(Qiagen, CA, USA) experimental evidence database.