Sample 35
Mus musculus
Protocols: Serum collected from heart following sacrifice. 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.
ENA first public
2017-12-12
ENA last update
2016-08-02
External Id
SAMEA4360782
INSDC center alias
University of Liverpool
INSDC center name
University of Liverpool
INSDC first public
2017-12-12T17:02:49Z
INSDC last update
2016-08-02T11:33:54Z
INSDC status
public
Submitter Id
E-MTAB-4878:Sample 35
broker name
ArrayExpress
common name
house mouse
disease
osteoarthritis
organism part
Serum
sample name
E-MTAB-4878:Sample 35
strain
C57BL6/J
treatment
destabilization of the medial meniscus