title: Sample 3
organism: Mus musculus
description: Protocols: 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 young normal donor 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: ENA first public
attributes.value: 2017-12-12
attributes.tag: ENA last update
attributes.value: 2016-08-02
attributes.tag: External Id
attributes.value: SAMEA4360776
attributes.tag: INSDC center alias
attributes.value: University of Liverpool
attributes.tag: INSDC center name
attributes.value: University of Liverpool
attributes.tag: INSDC first public
attributes.value: 2017-12-12T17:02:49Z
attributes.tag: INSDC last update
attributes.value: 2016-08-02T11:33:54Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-4878:Sample 3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: Young
attributes.tag: organism part
attributes.value: Joint
attributes.tag: sample name
attributes.value: E-MTAB-4878:Sample 3
attributes.tag: strain
attributes.value: C57BL6/J