Genomewide Analysis of mRNA Processing in Yeast Using Splicing-Specific Microarrays
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Supplementary on Science Online at www.sciencemag.org/cgi/content/full/296/5569/907/DC1. Microarray data are available at the Gene Expression Omnibus (GEO) at www.ncbi.nlm.nih.gov/geo/ under accession numbers GSE34 and GSE35.
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Fluorescently labeled target sequence sample preparation and hybridization were performed as described (5) by using 20 μg of total RNA primed with a mixture of oligo(dT) and random hexamers (7). Arrays were scanned and analyzed by using a GenePix 4000A scanner and GenePix Pro 3.0 software from Axon Instruments (Union City CA).
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The splice junction (SJ) index is the ratio of the mutant/wild-type ratios derived from the normalized signals from the splice junction probe to the exon 2 probe: SJ index = SJ mut /SJ wt divided by E2 mut /E2 wt obtained by subtracting the log 2 ratio of the exon 2 probe from the log 2 ratio of the splice junction probe. The intron accumulation (IA) index is obtained by subtracting the log 2 ratio of the exon 2 probe from the log 2 ratio of the intron probe. Because probe performance may not be directly related to absolute transcript amount these indexes depend idiosyncratically on the sequences of the probes. We also calculated the precursor/mature (PM) index which is obtained by subtracting the log 2 ratio of the splice junction probe from the log 2 ratio of the intron probe. This index mimics the unspliced/spliced ratio used in classical splicing studies (15).
Zhang and Schwer define the brp-to–3′ ss distance starting from 2 bases downstream of the brp adenosine to the Y of the 3′ ss YAG sequence. Therefore their distance of 12 nt corresponds to 17 nt from the actual branched A to the 3′ splice site G.
T. A. Clark C. W. Sugnet M. Ares Jr. unpublished observations.
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Thanks go to H. Igel for technical assistance; and to J. DeRisi T. Ferea T. Furey G. Hartzog D. Haussler H. Heynecker R. Perriman T. Powers L. Shiue and R. Samaha for advice. Funding was provided by a W. M. Keck Foundation Grant to the Center for Molecular Biology of RNA by National Cancer Institute grant CA77813 to M.A. and D. Haussler and by NIH grant GM40478 to M.A. T.C. was supported by a University of California Biotechnology training grant and an NIH Training Grant. C.S. is a Predoctoral Fellow of the Howard Hughes Medical Institute.