Rnase easy columns

RNA was extracted using hot phenol and purified over RNase easy columns (Qiagen) as previously described 64. A 15‐μg aliquot of total RNA was prepared for microarray analysis using the Superscript Plus Direct labelling kit (Invitrogen, Life Technologies), in two biological repeats with dye swaps. Paired samples were hybridised over two 44K Custom microarrays (Agilent) overnight at 65°C, scanned using GenePix 4000B scanner (Axon Instruments) and analysed using Genepix Pro 6.0 software(Axon Instruments). In house normalisation, scripts were applied (to gpr files) and the data uploaded into Genespring 7.3.1 software for analysis. Microarray data can be accessed at ArrayExpress accession E‐MTAB‐3455.

RNA was extracted using hot phenol and purified over RNase easy columns (QIAGEN, Valencia, CA) as previously described (Lyne et al. 2003 (link)). For strand-specific RT-PCR, one primer complementary to the sense or antisense transcript was added during first strand cDNA synthesis, while the second primer was added prior to the PCR amplification steps. cDNA for quantitative PCR (qPCR) was made using a Superscript II kit (Invitrogen, Carlsbad, CA). qPCR reactions were performed using a LightCycler 2.0 PCR system (Roche Diagnostics, Indianapolis, IN) and SYBR Green mix (Molecular Probes, Eugene, OR) using the appropriate primers.