Total RNA was extracted from whole embryos ground in liquid nitrogen using Trisol reagent. RNA was then treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.and purified using an RNAeasy kit (Qiagen). First-strand cDNA synthesis was performed using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 ug of starting RNA material. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) heated to 70C for 10 min and cooled to 15C after which 5x Superscript II First Strand buffer (Invitrogen), DTT ( 10 mM) and dNTPs ( 0.5 mM) were added. Superscript II RTase was added after a 20 min incubation (200Units/ug of RNA; Invitrogen) followed by a 20 min ramp to 42C and 60 min incubation at 42C. SuperScript II was inactivated at 75C for 15 min. Second-strand cDNA was synthesized by addition of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAseH and 0.2mM dNTPs to the first-strand synthesis reaction at 16C for 2 hours (all reagents were from Invitrogen). Double-stranded cDNA is treated with RNAseH (Epicentre) and RNAses A/T1 (Ambion), extracted using QIAquick PCR purification kit (Qiagen) and subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre, size distribution of fragmented DNA was verified on a 2% agarose gel) . The fragmented cDNA was then end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1x TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the microarrays for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Data processing
RNA abundance was estimated using a Wilcoxon Sign Rank Scan Statistic where the median of all pairwise average values of PM-MM is calculated for all probe pairs within a sliding window of 101bp of CHR_POS using quantile normalizing replicate arrays whose median array intensity was scaled to 38.
Expression profile of the first 24 hrs of Drosophila embryonic development using tiling arrays
Data table header descriptions
ID_REF
CHR_POS: chromosome_genomic position
VALUE
'signal' an estimate of RNA abundance using a Wilcoxon Sign Rank Scan Statistic where the median of all pairwise average values of PM-MM is calculated for all probe pairs within a sliding window of 101bp of CHR_POS using quantile normalizing replicate arrays whose median array intensity was scaled to 38.
ABS_CALL
the call in an absolute analysis that indicates if at CHR_POS the 'signal' is significantly over background levels with P indicating RNA is present and A indicating absent. This call was produced by applying a 'signal' cutoff associated with a 5% false positive rate in negative bacterial controls tiled on the array and calling all probes P within 50bp of probes which pass the threshold. If the length of a stretch of probes called P is less than 90bp, they are re-classified as A.
