title: Illumina HiSeq 2000 paired end sequencing; Small non-coding RNAs as biomarkers of musculoskeletal ageing and osteoarthritis
design: Small non-coding RNAs as biomarkers of musculoskeletal ageing and osteoarthritis
library_construction_protocol: Skin removed from hind legs and stifle joints collected. Approximately 4mm each side of the joint was collected with no muscle included.  destabilization of the medial meniscus (DMM)  model of traumatic osteoarthritis 100ng of small RNA was added in a volume of 6ul together with 1ul of 3'SR adapter (diluted 1+2). This was heated at 70 degrees for 2minutes and cooled on ice. Ligation buffer and enzyme were added and the reaction incubated for 1 hour at 25 degrees. 2ul of Tobacco Acid Pyrophosphatase (TAP)(Epicentre) 5u/ul was added to each reaction and incubated at 37 degrees for 1 hour. Tobacco Acid Pyrophosphatase (TAP) hydrolyzes the phosphoric acid anhydride bonds in the triphosphate bridge of the cap structure found in most eukaryotic mRNA, releasing the cap nucleoside and generating a 5 ́-monophosphorylated terminus on the RNA molecule. This is inactivated in the subsequent step which heats the samples to 75 degrees for 5 minutes The samples were hybridized to the Reverse Transcription Primer at a dilution of 1+2 from the supplied stock of primer. This step is important to prevent adaptor-dimer formation. The SR RT Primer hybridizes to the excess of the 3 ́ SR Adaptor (that remains free after the 3 ́ ligation reaction) and transforms the single stranded DNA adaptor into a double-stranded DNA molecule. dsDNAs are not substrates for ligation mediated by T4 RNA Ligase 1 and therefore do not ligate to the 5 ́ SR Adaptor in the subsequent ligation step. Following this 5”ligation for 1 hour at 25 degrees, a reverse transcription was carried out at 50 degrees for 1 hour using ProtoScript II Reverse Transcriptase. The samples were then amplified for 15 cycles and incorporating a barcode as part of the priming process.  The samples were mixed into 3 pools,(mixing across groups) and size selected on a Sage Pippin cassette 2% DF marker E using a size range 120-300bp. The size selected material was purified with Ampure beads(1:1) (Agencourt). The pools were checked by Qubit analysis and Bioanalyser High Sensitivity chip.
attributes.tag: Experimental Factor: N/A
attributes.value: developmental stage
attributes.tag: Experimental Factor: osteoarthritis
attributes.value: disease
attributes.tag: Experimental Factor: Joint
attributes.value: organism part
attributes.tag: Experimental Factor: destabilization of the medial meniscus
attributes.value: treatment