title: Illumina HiSeq 2500 paired end sequencing; RNA-seq of laser micro-dissected cartilage after surgical induction of osteoarthritis in increased ER stress mice
design: RNA-seq of laser micro-dissected cartilage after surgical induction of osteoarthritis in increased ER stress mice
library_construction_protocol: Non-calcified articular cartilage was collected from the sections from sham‐operated and DMM‐operated mice using laser-capture microdissection (Arcturus Bioscience). Osteoarthritis was induced in 10-12 week old male mice by DMM of the right knee. The left knee was the non-operated control. Sham operations were performed in independent animals where the medial meniscotibial ligament was visualised but not transected. Total RNA was extracted from pooled laser-microdissected sections from each individual mouse joint using TRIzol reagent according to the specifications of the manufacturer (Invitrogen). Each mouse articular cartilage yielded ~20-30ng of total RNA. The integrity of total RNA was assessed on the Agilent Technology Bioanalyzer 2100 using RNA6000 Pico-Chip (Agilent) and quantified using a QuBit assay (Life Technologies). RNA sequencing cDNA libraries were generated using the NuGen Ovation® Single Cell RNA-Seq system (NuGEN Technologies Inc., Part No.0342) according to manufacturer's instructions. Adapter indices were used to multiplex libraries, which were pooled prior to cluster generation using a cBot instrument.
attributes.tag: Experimental Factor: genotype
attributes.value: ColIITg cog
attributes.tag: Experimental Factor: disease
attributes.value: osteoarthritis, knee
attributes.tag: Experimental Factor: injury
attributes.value: right knee destabilisation of the medial meniscus