strain background: mixed between SvJ/129 and Black Swiss (Taconic) genotype/variation: wild type (WT) age: 2-3 days, pups tissue: Skeletal muscle developmental stages: Proliferating primary myoblasts infected with: adenovirus expressing Ankrd2 or control GFP
Treatment protocol
Growing myoblasts were infected with adenoviruses expressing Ankrd2 (Ad-Ankrd2) or GFP (Ad-GFP) 20h before collection (Stage1: proliferating myoblasts). When the confluence went above 80%, the differentiation medium was added to the proliferating myoblasts (normally day 2 in the morning) for 24h (Stage2: Fusing myoblasts) or 96h (Stage3: Differentiated myoblasts). For both stage 2 and stage 3 samples Ad-Ankrd2 or Ad-GFP were added 20h before collection. Two kind’s of media were used: 1) Proliferation medium: 20% FBS,1% Penicillin-Streptomycin,1% Glutamine,DMEM with 4500 g/l glucose. 2) Differentiation medium: 2% Horse Serum, 1% Penicillin-Streptomycin,1% Glutamine,DMEM with 4500 g/l glucose. Cultures were incubated at 37 ºC in a humidified incubator with 5% CO2.
Growth protocol
The pups of both Ankrd2-KO and WT mice were sacrificed at day 2-3. The limbs were dissected by removing the skin and pulling the arms and legs out of their sockets. The tissues were minced by cutting into small pieces and were digested by 5-6 rounds of trypsin incubation/centrifugation.The surnatants containing mostly blood cells were discarded. The cell mixtures were triturated and the fat cells from the higher phase were removed. The remaining cells, myoblasts, were filtered and plated.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated with TRIzol reagent (Invitrogen) following the standard protocol. The Nanodrop ND-1000 was used for RNA quantification and RNA 6000 Nano LabChip kit (Agilent Technologies) was used for RNA quality control in conjunction with an Agilent Bioanalyzer 2001.(Agilent Technologies) was used for RNA quality control in conjunction with an Agilent Bioanalyzer 2001.
Label
Cy3
Label protocol
50 ng RNA was amplified and labeled using the Agilent’s Low Input Quick Amp WT Labeling Kit, one-color, (catalogue number 5190-2943), following the Manufacturer’s instructions. 2.3 µl of RNA sample was mixed to 1 µl WT Primer, 2 µl Agilent One-Color RNA Spike-In (1:20,000 dilution, catalogue number 5188-5282) and denatured for 10 min at 65 ºC. First Strand Buffer, DTT, dNTP mix, and AffinityScript RNaseBlock mix were added and cDNA synthesis was carried out for 2 h at 40 ºC. After 15 min at 70 ºC to inactivate the enzymes, complementary RNA (cRNA) was generated using T7 RNA polymerase, which simultaneously amplifies target material and incorporates Cy3-labeled CTP. Run-off in vitro transcription reactions proceeded for 2 h at 40 °C. RNeasy Mini spin columns (Qiagen) were used to further purify the labeled-amplified cRNA samples.
Hybridization protocol
600 ng of Cy3-labeled, linearly amplified cRNA was mixed with 5 µl of 10X Blocking Agent, 1 µl of 25X Fragmentation Buffer, and water of Agilent Hybridization Kit (catalogue number 5188-5242) to a final volume of 25 µl. Reaction was incubated at 60°C for exactly 30 min to fragment cRNA. 40 µl of hybridization mix, composed by equal volumes of fragmented cRNA and 2X GEx Hybridization Buffer HI-RPM, was dispensed onto one of the 8 arrays of the Agilent SurePrint G3 Mouse GE 8x60K Microarrays (catalogue number G4852A). The full microarray slide was loaded into an Agilent SureHyb chamber and hybridization was performed in a hybridization oven at 65°C for 17 h. Rotation was set at 10 rpm. At the end of hybridization, slides were washed for 1 min at 22C in Wash Solution 1 (catalogue number 5188-5325) and 1 min in Wash Solution 2, pre-warmed to 37 C (catalogue number 5188-5326).
Scan protocol
Microarrays were scanned using the Agilent Technologies Scanner G2505C US22502723 (ChipScan software version A.8.5.1), scan region 61 X 21.6 mm, scan resolution 3 micron single pass, Green PTM 100%, no extended dynamic range (NO XDR).
Description
SAMPLE 4 Gene expression after 20h Ad-Ankrd2 infection in WT proliferating myoblasts
Data processing
Data was extracted from the scanned image using the Agilent Feature Extraction Software version 10.7.3.1 (protocol GE1_107_Sep09). Quantile inter-arrays normalization was performed with the Expander software (Bioinformatics 2003; 19:1787-99) on the gProcessedSignal column of the result table and normalized data is reported in the VALUE column. Only characterized lincRNAs (with the “accession string” field of platform defined with a symbol) was used in the normalization process. The other lincRNAs (with the 'accession string' field of platform defined with the chromosomal location) was not used in the normalization process and marked as “null”. Probes with gIsPosAndSignif value of zero were disregarded and labeled as 'null'. Mean values were calculated for multiple probes with the same Agilent Probe ID.
