normalization data transformation protocol
Gene expression data were processed using the lumi package in R. Probes were filtered to remove those where the detection p-value (representing the confidence that expression is above the background of the negative control probe) was greater than 0.01 in all samples. Expression data were transformed to the log2 scale, then normalised using quantile normalisation. Comparisons between the DMSO-treated and inhibitor-treated samples were performed using the R package limma. P-values were adjusted to control the false discovery rate by using the Benjamini and Hochberg multiple testing correction.
array scanning protocol
Hybridised Illumina HT12 v4 arrays were scanned using an Illumina BeadArray Reader.
nucleic acid hybridization to array protocol
Labeled RNA extracts were hybridized to Illumina HT12 v4 BeadArrays using the Illumina Direct Hybridisation Assay.
nucleic acid labeling protocol
Total RNA was extracted and labelled using the Illumina TotalPrep RNA Amplification kit (Ambion).
Human erythroleukemia (HEL) cell lines were maintained in standard RPMI 1640 medium, supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin/glutamine (GIBCO, Invitrogen). Cultures were incubated at 37C in the presence of 5% CO2 and were passaged 1:6 every 2-3 days.
The compounds used in this study were: TG101209, an inhibitor of Janus kinase 2 (JAK2), commercially available from TargeGen Inc. and the BET inhibitor: GSK1210151A (I-BET151) which was kindly provided by GlaxoSmithKline. HEL cells were adjusted in media to 1e6 cells/ml and treated for 4 hours with 1 uM final concentration of either: TG101209, I-BET151 or equivalent volume of DMSO (vehicle).
nucleic acid extraction protocol
Equal numbers of cells receiving each treatment were washed in 1x ice-cold PBS and lysed in 600 ul of RLT buffer (RNAeasy kit, Qiagen) supplemented with beta-mercaptoethanol (beta-ME). One volume (600 ul) of 70% ethanol was added to all samples and mixed by pipetting; mRNA was further purified with the RNAeasy kit, following the manufacturer's instructions.