Fig. 2

RNA-SEQ analysis of PBMLs isolated from RA patients and healthy subjects at 6 am and 6 pm. a, b Mean-difference (MD) plots for gene expression differences between RA (n = 10) and healthy (n = 9) subjects at a 6 am and b 6 pm. c, d MD plots showing differences by time of day within each group, c RA and d healthy subjects (using the model ~time + subject). Logarithm of counts per million reads (LogCPM) was calculated for each subject, and the mean line is shown in black. e Expression of the circadian clock gene PER3 was determined from RNA-SEQ data. Some genes only changed by time of day in RA, f ORCL. Comparisons were made using Edge R glmLRT, and exact p values are shown (FDR corrected). Differentially expressed genes between healthy and RA at 6 am were tested for enrichment using the PANTHER/Reactome database (a selection is shown), genes that were lower in RA at 6 am (g) or higher in RA at 6 am (h). i Reactome pathway analysis of the toll-like receptor 4 (TLR4) cascade network discovered in h is represented. Green showing 6 am upregulated genes, specifically of the TLR signalling pathway (j) The innate immune system genes were analysed by iRegulon to identify enriched transcription factor binding, based on the TRANSFAC database and ENCODE (using iRegulon). Normalised enrichment scores (NES) indicate a motif that covers a large proportion of the input genes (> 3, which corresponds to an FDR between 3 and 9%)