Comment[ArrayExpressAccession]	E-MTAB-1692
MAGE-TAB Version	1.1
Investigation Title	Inflammatory response in primary muscle cell cultures in Atlantic salmon (Salmo salar)
Comment[Submitted Name]	Inflammatory response in primary muscle cell cultures in Atlantic salmon (Salmo salar)
Experiment Description	This experiment was aimed at understanding the transcriptomic response of primary muscle cell cultures to 24h rIL-1beta stimulation
Experimental Design	stimulus or stress design	in_vitro_design	co-expression_design	compound treatment design
Comment[AEExperimentType]	transcription profiling by array
Comment[AEExperimentDisplayName]	Transcription profiling by array of primary muscle cell cultures from Atlantic salmon (Salmo salar) treated with rIL-1beta
Experimental Factor Name	chemical compound
Experimental Factor Type	chemical compound
Quality Control Type	biological replicate
Quality Control Term Source REF	EFO
Public Release Date	2013-12-04
Person Last Name	Martin
Person First Name	Sam
Person Mid Initials	AM
Person Email	sam.martin@abdn.ac.uk
Person Phone
Person Address	School of Biological Sciences, University of Aberdeen, Aberdeen, AB24 2TZ, UK
Person Affiliation	University of Aberdeen
Person Roles	submitter
PubMed ID	24180744
Publication Author List	Pooley NJ, Tacchi L, Secombes CJ, Martin SA
Publication Title	Inflammatory responses in primary muscle cell cultures in Atlantic salmon (Salmo salar)
Publication Status	in preparation
Protocol Name	P-MTAB-33128	P-MTAB-33129	P-MTAB-33130	P-MTAB-33131	P-MTAB-33132	P-MTAB-33133
Protocol Type	growth protocol	treatment protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning protocol
Protocol Description	Atlantic salmon (mean weight of 25g and mean length of 12cm) were used for skeletal muscle myosatellite cell extraction to produce 12 wells for biological replication using a modification of the protocol described in Matschak TW, Stickland NC: The growth of Atlantic salmon (Salmo salar L.) myosatellite cells in culture at two different temperatures. Experientia 1995, 51:260?266 	Cells were grown for 4 days post extraction before stimulation with either 25ng/ml of recombinant IL-1beta (rIL-1beta) or PBS for control	RNA was extracted from each well of two 6-well plates using RNAeasy extraction kit (Qiagen) according to manufacturers instructions and resuspended in 50 ul RNase free H2O. The concentration was determined by spectrophotometry (NanoDrop, ND1000, Lab Tech) and the integrity of the RNA was determined by electrophoresis (Agilent Bioanalyser 2100). The RNA was then stored at -80C until required. Antisense amplified RNA (aRNA) was produced from 1ug each total RNA isolation using the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion, Applied Biosystems).  These aRNA samples were quantified and quality assessed (Nanodrop spectrometry) and stored at -80C until required.	Cy dye suspensions (Cy3 & Cy5) in sufficient quantity for all labelling reactions were prepared by adding 45 uL high purity dimethyl sulphoxide (Stratagene) to each tube of Cy dye (dye Cy3TM or Cy5TM mono-reactive dye pack, Amersham). To attach the Cy dyes, 3 ug each aRNA sample was suspended in 6 uL nuclease-free H2O and heated to 70C for 2 min. When cooled to room temperature, 2 uL of coupling buffer (0.5 M NaHCO3; pH 9.2) and 2 uL of Cy3 dye suspension stock was added and then incubated for 1 h at 25C in the dark.  For labelling the common pooled reference sample with Cy5, a scaled-up batch reaction was similarly performed. Unincorporated dye was removed by column purification (DyeExTM 2.0 spin kit QIAGEN). Dye incorporation and aRNA yield were quantified by spectrophotometry (NanoDrop ND-1000).	For the hybridisation 825ng of each labelled template was fragmented in the presence of 11ul of 10X blocking agent, 2.2ul of 25X Fragmentation buffer (Agilent), and made up to a final volume of 20ul with nuclease-free dH2O. Fragmentation progressed in the dark at 60C for 30 min and then 57ul of 2X GEx Hybridisation buffer (Agilent) was added to each sample and 103ul of each hybridisation solution was dispensed onto the Agilent 4x44K Atlantic salmon Salmo salar2 oligo array . The hybridisations were performed in a Microarray Hybridisation Oven (Agilent) overnight (18 h) at 65C. Following hybridisation, the slides were rinsed in Gene Expression Wash buffers 1 and 2 (Agilent) following the manufacturers instructions	Processed microarrays were scanned using a GenePix personal 4100A Scanner (Axon Instruments) at a resolution of 5um and saved as *.TIF files. The PMT for each channel was adjusted such that less than 0.1 % of features were saturated and that the mean intensity ratio of the Cy3 and Cy5 signals was close to one.
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO	EFO
Term Source Name	EFO	ArrayExpress
Term Source File	http://www.ebi.ac.uk/efo	http://www.ebi.ac.uk/arrayexpress
SDRF File	E-MTAB-1692.sdrf.txt
Publication DOI	10.1186/1471-2164-14-747