Immunoprecipitation (IP) protocol of endogenous mRNA-HuR complexes was performed as described by (Jayaseelan et al., 2014; Keene et al., 2006). For RIP-ChIP analyses, frozen tissue samples from human cancer panel (n=8 neurofibroma and n=12 for MPNST) were homogenized in polysome lysis buffer [100 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7.0, 0.5% NP-40, 1 mM DTT, 100 units/ml RNase OUT, 1X Protease Inhibitor Cocktail), incubated for 30 min on ice, and centrifuged at 13 000 rpm, 4ºC for 30 minutes. 500 l of lysates were pre-cleared by incubating with 25 l of protein A-sepharose 4B beads (Merck) and anti-mouse IgG (BD Biosciences) in 1 ml of NT2 buffer [50 mM Tris pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40] and incubating under rotation for 30 min. The pre-cleared extracts were then divided and incubated with 50 l of protein A-sepharose 4B beads, pre-coated with anti-HuR or anti-mouse IgG antibodies. After incubation, beads were washed 5 times with 1 ml NT2 buffer and bound RNA recovered after proteinase K digestion (Roche) and phenol chloroform extraction. RNAs were then submitted to the Genomics Analysis Platform at CIC bioGUNE for analysis on HUMAN HT-12 V4 arrays (Illumina).
Growth protocol
Frozen tissue samples from human cancer panel (n=8 neurofibroma and n=12 for MPNST) were homogenized in polysome lysis buffer [100 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7.0, 0.5% NP-40, 1 mM DTT, 100 units/ml RNase OUT, 1X Protease Inhibitor Cocktail), incubated for 30 min on ice, and centrifuged at 13 000 rpm, 4ºC for 30 minutes.
Extracted molecule
total RNA
Extraction protocol
Phenol chloroform extraction
Label
Biotin
Label protocol
TargetAmp™ Nano-g™ Biotin-aRNA Labeling Kit for the Illumina® System (Epicentre)
Hybridization protocol
Standard Illumina hybridization protocol
Scan protocol
Standard Illumina scanning protocol
Description
IgG IP_sMPNST3
Data processing
The data were normalised using quantile normalisation with lumi in R After background correction with GenomeStudio and log2 transformation, probes with a detection p-value greater than 0.01 for all samples were discarded, using the detectionCall function in the lumi R package.
