Illumina HiSeq 2000 paired end sequencing; RNA-Seq analysis of human intact and damaged osteoarthrtitic cartilage following total knee replacement.
RNA-Seq analysis of human intact and damaged osteoarthrtitic cartilage following total knee replacement.
Cartilage was obtained under Ethics Committee approval with written informed consent from 8 patients with symptomatic OA at total knee replacement (n=8, age range 65-79 years, mean age 70.3). Cartilage from paired osteochondral samples were isolated from the intact PLC (posterior lateral condyle) and the damaged DMC (distal medial condyle) for RNA-seq analysis. Strand specific RNA-seq libraries were generated from 0.5-1 g RNA using the TruSeq Stranded mRNA assay (Illumina, Inc.) using the manufacturers Stranded mRNA Sample Preparation Kit and 101bp paired-end reads were generated with each  sample yielding at least 39 million total reads. Total RNA was extracted from 200-400 mg of cartilage using TRIzol (Life-technologies) reagent and homogenisation (Braun Mikrodismembrator) following freezing in liquid nitrogen. The RNA was purified using RNeasy Qiagen clean-up columns (Qiagen) and for sequencing had a RIN score of >6 (2200 TapeStation, Agilent Technologies).
Experimental Factor: Damaged
phenotype
Experimental Factor: 67
age
Experimental Factor: male
sex