Cindy Boer

186 | Chapter 4.1 RNA sequencing and proteomic analysis of the UK cohort Full methods have been described previously[90, 91]. Briefly: DNA, RNA, and protein was extracted from matched intact and degraded cartilage samples from 38 patients undergoing total joint replacement surgery: 29 knee and 9 hip osteoarthritis patients. All patients provided full written informed consent before participation. The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an Institutional Review Board (IRB)- or Eth- ics Committee (EC)-approved protocol. Proteomics analysis was performed on intact and degraded cartilage samples from 24 individuals and gene expression analysis on samples from all 38 patients. For the proteomics we performed LC-MS analysis on the Dionex Ultimate 3000 UHPLC system coupled with the Orbitrap Fusion Tribrid Mass Spectrometer. Abundance values were normalised by the sum of all protein abundances in a given sample, then log2-transformed and quantile normalised. We restricted the analysis to 3,917 proteins that were quantified in all samples. Differential abundance was performed using a within-individual paired sample design in limma in R[92]. RNA sequencing was performed on the Illumina HiSeq 2000 (75bp paired-end read length). We determined the gene-level counts from transcript-level quantification using salm- on 0.8.219 with GRCh38 cDNA assembly release 87 downloaded from Ensembl. Lim- ma-voom[93] was used to remove heteroscedasticity from the estimated expression data. We tested genes for differential expression using limma in R (with lmFit and eBayes), based on a within-individual paired sample design. For the proteomics and RNA sequencing significance was defined at 5% Benjamini-Hochberg FDR to correct for multiple testing ( Supplementary Table 7 ). Transcriptomics and analysis in subchondral bone in the Taiwan OA cohort Knee joints samples were collected from 11 Han Chinese patients underwent total knee replacement surgery (TKR). The subchondral bone tissues underneath the intact and eroded cartilage were obtained as previously described[94]. RNA was extracted as de- scribed[95]. RNA (400ng) per sample were used for cRNA synthesis and amplification. Cyanine 3-labeled cRNA was then purified and hybridized to Agilent whole human ge- nome 44 k microarray chips (Agilent Technologies) according to the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA). The array signal intensities were analysed by the Agilent GeneSpring GX software (version 11.5; Agilent Technologies). Gene expression values were normalized using quantile normalization; probes with low signal intensities were excluded by setting the filter above 32. The normalized values were log transformed and compared using t test. Differentially expressed genes were defined at ≥2 fold-change with Benjamini-Hochberg corrected P-value ≤0.05.