To generate Tollrm9/Tollrm10 females, females of the genotype Tollrm10/TM3 Sb were mated with males of the genotype Tollrm9/TM3 Ser. Non-Sb, non-Ser females of the genotype Tollrm9/Tollrm10 were selected.
To generate pipe-/pipe- females, pipe664/TM3 Sb females were mated with males of the genotype pipe386/TM3 Sb. Non-Sb females of the genotype pipe386/pipe664 were selected.
Extracted molecule
total RNA
Label
bio-dideoxy ATP
Description
To generate pipe-/pipe- females, pipe664/TM3 Sb females were mated with males of the genotype pipe386/TM3 Sb. Non-Sb females of the genotype pipe386/pipe664 were selected. To generate Tollrm9/Tollrm10 females, females of the genotype Tollrm10/TM3 Sb were mated with males of the genotype Tollrm9/TM3 Ser. Non-Sb, non-Ser females of the genotype Tollrm9/Tollrm10 were selected. Females of the genotype Toll10B/+ were obtained directly from the balanced stock (Toll10b/TM3Sb Ser and Toll10b/OR60). Two hour embryo collections were collected from females of the selected genotypes, either pipe386/pipe664, Tollrm9/Tollrm10,or Toll10B/+, on apple juice/yeast plates at 25 degrees celcius. The embryos were placed for an additional 2 hrs at 25 degrees celcius to obtain samples of embryos aged 2-4 hrs. Embryos were collected for five different days, dechorionated, and frozen in liquid nitrogen for storage at -80 degrees celcius until RNA was to be isolated. The different collections were pooled for each sample in order to better normalize the age of these embryo populations. Once sufficient amounts of embryos had been collected (~500 embryos), total RNA was extracted from them using Trizol Reagent (GIBCO-BRL) according to the manufacturer's protocol. For each sample, 100 ug of total RNA was further purified using the RNeasy Mini Kit (Qiagen) following the Rneasy Mini Protocol for RNA cleanup. Total RNA was prepared independently three times from embryos of each genetic background. First-strand cDNA synthesis was performed by using SuperScript II reverse transcriptase in the reaction volume of 105 ul for 15 ug of starting RNA material. The RNA was mixed with random hexamers (83.3 ng/g mRNA), heated to 70 degrees celcius for 10 min, and cooled to 15 degrees celcius after which 5x SuperScript II First Strand buffer, DTT (10 mM), and dNTPs (0.5 mM) were added. SuperScript II was added after a 20-min incubation (200 units/ug RNA) followed by a 20-min ramp to 42 degrees celcius and 60-min incubation at 42 degrees celcius. SuperScript II was inactivated at 75 degrees celcius for 15 min. The second-strand cDNA was synthesized by addition of 50 units of Escherichia coli DNA ligase, 200 units of E. coli DNA polymerase I, 10 units of E. coli RNase H, and 0.2 mM dNTPs to the first-strand synthesis reaction at 16 degrees celcius for 2 hrs. Double-stranded cDNA was treated with RNase H (Epicentre Technologies) and RNases A/T1 (Ambion), extracted by using a QIAquick PCR purification kit (Qiagen), and subjected to further fragmentation to 50-100 bp by DNase I (1 unit/ul; Epicentre Technologies; size distribution of fragmented DNA was verified on a 2% agarose gel). The fragmented cDNA was then end-labeled with 70 nM bio-dideoxy ATP (PerkinElmer) by using 6-10 units of terminal deoxynucleotidyltransferase (TdT; Roche Diagnostics) per ug of fragmented DNA in 1x TdT buffer (Roche Diagnostics) and 5 mM CoCl2 (Roche Diagnostics) for 2 hrs at 37 degrees celcius. The labeled DNA material was subsequently hybridized to Affymetrix Drosophila-tiled genomic microarrays for 18 hrs at 45 degrees celcius in a 3 M tetramethyl ammonium chlorate/1x Mes-based solution. All reagents were from Invitrogen, except where noted otherwise.
Data processing
Processing of the microarray data was performed in three basic steps using TiMAT (http://bdtnp.lbl.gov/TiMAT/): data normalization, sliding window summary statistics, and enriched region identification. To normalize the data, all cel files were grouped together and the perfect match intensities(PM) were quantile-normalized and median-scaled to 100. Mismatch intensities were discarded. To identify regions enriched relative to each other, all pair-wise comparisons were made between pipe, Tollrm9/Tollrm10, and Toll10B data (i.e. pipe vs. rm9/rm10, pipe vs. 10B, rm9/rm10 vs. 10B, rm9/rm10 vs. pipe, 10B vs. rm9/rm10, and 10B vs. pipe). Cel files for a particular pairing were divided into treatment and control. Their intensities were mapped to the genome and a ratio score was calculated for each oligo by dividing the average treatment by the average control. To minimize noise, a sliding window of 675bp, containing approximately 19 oligos, was advanced, one oligo at a time across each chromosome (similar results were obtained using a window of 250bp containing 7 oligos). A trimmed mean of the grouped oligo ratios was used to score each window. To collapse overlapping windows into enriched regions, windows that (i) intersect by greater than 100bp, (ii) exceed a low threshold of 1.25x, and (iii) contain more than 5 oligos were joined.
Comprehensive identification of Drosophila dorsal-ventral patterning genes using a whole-genome tiling array
Data table header descriptions
ID_REF
CHR_POS: chromosome_genomic position
VALUE
MEDIAN_RATIO: An enrichment score (Median Fold Difference; MFD) for each interval was calculated by identifying the best 225bp sub window within the interval based on the median of the associated oligo ratio scores. The intervals were ranked using this enrichment score
TRIMMED_MEAN_RATIO
Highest scoring trimmed mean ratio window in the interval
WILCOXON_RANK_SUM
Wilcoxon rank sum test (-10Log10(p-value))
ABS_CALL
Probes that fall within an enriched region are marked as present with a "P" and the rest are noted as absent
