For the expression analysis, MLP-29 cells were progressively starved for 1 week, and subsequently stimulated with HGF, or control medium, for 1, 6 and 24 hours in the presence of 2% bovine serum (BS; Sigma). The time-course stimulation was organized so that cells from all the experimental points could be lysed at the same time, to eliminate any possible variability due to culture conditions, cell density, time from the last passage, medium exhaustion. Stimulations were performed with recombinant HGF. The experiment was repeated twice, to generate biological replicates.
Growth protocol
MLP-29 are grown in DMEM with 10% bovine serum
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using the Trizol Plus purification Kit (Invitrogen, Frederick, MD, USA, cat. no.12183555), according to the manufacturer’s protocol. Quantification and quality analysis of RNA was performed on a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA).
Label
Biotin
Label protocol
Synthesis of cDNA and biotinylated cRNA were performed using the Illumina Total-Prep RNA Amplification Kit (Ambion, Austin, TX, USA, cat. no. IL1791), according to the manufacturer’s protocol, for which hybridization was carried out on Illumina Mouse8_V2 arrays
Hybridization protocol
0.85 microgram of biotin-labeled cRNA was hybridized to Illumina MurineRef8 V2 at 55°C overnight. BeadChips were incubated with Cy3 streptavidin and washed according to the manufacturer’s protocol.
Scan protocol
The hybridized BeadChips were scanned by Illumina BeadScan confocal scanner and analyzed by Illumina's GenomeStudio software.
Description
HGF6h-b
Data processing
Data were normalized by Illumina's GenomeStudio software (v.1.9.0) with cubic spline normalization
