Whole adult Drosophila melanogaster dsx[swe]/Df(3R)dsx[M+15] and tra2B/Df(2R)trix flies were grown at 25C on PB medium (KD Medical, Columbia, MD) for 5 to 7 days post eclosion. Dissected ovaries were quick frozen on dry ice and total RNA was extracted (Andrews et al, Genome Research 10:2030-2043) using Trizol reagent (GibcoBRL, Gaithersburg, MD). Probes were labeled with Cy3 or Cy5. To synthesize probes, RNA was isolated by a single round of poly(A) selection using Oligotex resin (Qiagen, Valencia, CA). The purified mRNA was quantified using riboGreen dye (Molecular Probes) in a fluorescent assay. RiboGreen dye was diluted 1:200 (v/v final) and mixed with known RNA concentrations determined by absorbance at 260 nm) ranging from 1 to 10,000 ng/ml. Fluorescence was measured in 96-well plates with a FLUOstar fluorometer (BMG Lab Technologies, Germany) fitted with 485 nm (excitation) and 520 nm (emission) filters. Between 25 and 100 ng mRNA were separated on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), to examine the mRNA size distribution. 600 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using 50 mM Tris-HCl pH 8.3, 75 mM KCl, 15 mM MgCl2, 4 mM DTT, 2 mM dNTPs (0.5 mM each), 2 µg Cy3 or Cy5 random 9mer (Trilink, San Diego, CA), 20 U RNase inhibitor (Ambion), 200 U MMLV RNase H-free reverse transcriptase (Promega, Madison, WI) and mRNA. Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on a size exclusion column, concentrated by ethanol precipitation and resuspended in hybridization buffer. Probes were added between two subarray slides and developed as described (Yue et al.2001, NAR. 29:e41).
Data processing
Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA) at 535 nm. Signal intensities were initially captured using GenePix Pro 4.1 (Axon Instruments, Foster City CA). Normalization by within -slide print tip loess and between-slide quantile, along with subsequent analyses, were performed using the bioconductor package LIMMA (v.1.6.7) (Smyth et.al, 2004).
Global analysis of X chromosome dosage compensation
Data table header descriptions
ID_REF
Log2Cy3
Log2 transformed intensity signal from Cy3 channel. Values are normalized within each array by print tip Loess and across the series using quantile normalization in Bioconductor.
Log2Cy5
Log2 transformed intensity signal from Cy5 channel. Values are normalized within each array by print tip Loess and across the series using quantile normalization in Bioconductor.
VALUE
Log2 transformed ratio of corrected Cy3/Cy5 signal calculated by taking the corrected Log2Cy5 signal value and subtracting the Log2Cy3 signal value for each element
Cy3_SIGNAL
Raw median signal intensity data from from Cy3 channel acquired by Genepix
Cy5_SIGNAL
Raw median signal intensity data from from Cy5 channel acquired by Genepix
BCy3
Median background intensity signal from Cy3 channel acquired by Genepix
BCy5
Median background intensity signal from Cy5 channel acquired by Genepix
