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The Supplementary Material referred to in this article can be viewed at www.interscience.wiley.com/jpages/1058-8388/suppmat

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jws-dvdy.21211.fig1.tif147K Supplemental Figure 1. Microarray Probe Design and RNA PurificationA) Oligonucleotide probes containing 2 (44 nucleotides) or 3 (66 nucleotides) regions of complementarity to eithermiR-10b , ormiR-124awere coupled to mirror slides for initial microarray hybridization experiments. Individual antisense regions are indicated in green, red, or blue. B) Total RNA from zebrafish embryos was fractionated using either MirVana isolation kits or sucrose gradients. RNAs were separated on 12% 19:1 polyacrylamide gels and stained with ethidium bromide. MirVana fractions (Lanes 1, 2) and the top three sucrose gradient fractions (Lanes 3-5) are shown. Northern blot of the same RNAs in B using probes againstmiR-124a . C) Small RNAs isolated from 2 days post fertilization (2 dpf) zebrafish embryos were fluorescently labeled and hybridized to small scale arrays containing the probes described in A and pixel intensities were measured. Despite the increase in base pairing, no significant difference in fluorescence intensity was observed between the long and short oligonucleotides so that final arrays were printed with 2 regions of complementarity.
jws-dvdy.21211.fig2.tif214K Supplemental Figure 2. Microarray Sensitivity and SpecificityA,B) Sensitivity. Increasing amounts of a 22 nucleotide RNA encoding tomato lycopene synthase were spiked into microarray hybridizations and signal intensities were determined at each concentration after background and negative control subtraction. Values are shown in graphical (a) and heat map (b) format. As little as 1pg could be detected well above background. C) RNA Labeling. To optimize for the levels of RNA needed in each hybridization, signal intensities from 9 different miRNAs were determined after microarrays performed using from 0.1 to 4000ng labeled RNA. APC is a probe complementary to the zebrafish adenomatous polyposis coli mRNA. Based on these results, we chose to use 2ug for all subsequent arrays. D) Specificity. Single nucleotide specificity using Let-7 family members and mismatch controls. Four members of the let-7 miRNA family are shown with indicated single nucleotide differences. Two and six mismatches were also incorporated intomiR-124ashort oligonucleotides. E, F) Heat map representation of signal intensities for the miRNAs shown in D. Specificity is demonstrated by the lack of cross-hybridization between probes (Red: high expression values; Blue: low to zero expression values).
jws-dvdy.21211.fig3.tif308K Supplemental Figure 3. Northern blot VerificationMicroarray results were verified for three microRNAs using Northern blots.
jws-dvdy.21211.fig4.tif430K Supplemental Figure 4. Detailed Cyclopamine Expression Profiles Detailed view of expression analysis shown in Figure 3. Blue indicates little to no expression and red indicates high expression.
jws-dvdy.21211.fig5.tif427K Supplemental Figure 5. Detailed DAPT Expression ProfilesDetailed view of expression analysis shown in Figure 4. Blue indicates little to no expression and red indicates high expression.
jws-dvdy.21211.tbl1.xls186K Supplemental Table 1. Detailed Array DesignExact array design and probe sequences for all spots on the array as per MIAME guidelines.
jws-dvdy.21211.tbl2.xls1472K Supplemental Table 2. Raw Array Data During Wild Type Zebrafish DevelopmentF635 Median is the median intensity level of the signal. B635 Median is the median intensity level of the background surrounding the probe. A flag of -50 means that the signal could not easily be distinguished from background, and a flag of 0 means that the signal is present. Each stage is presented on a different sheet within the same excel workbook.
jws-dvdy.21211.tbl3.xls720K Supplemental Table 3. Raw Array Data After Cyclopamine TreatmentF635 Median is the median intensity level of the signal. B635 Median is the median intensity level of the background surrounding the probe. A flag of -50 means that the signal could not easily be distinguished from background, and a flag of 0 means that the signal is present. Each stage is presented on a different sheet within the same excel workbook.
jws-dvdy.21211.tbl4.xls710K Supplemental Table 4. Raw Array Data After DAPT TreatmentF635 Median is the median intensity level of the signal. B635 Median is the median intensity level of the background surrounding the probe. A flag of -50 means that the signal could not easily be distinguished from background, and a flag of 0 means that the signal is present. Each stage is presented on a different sheet within the same excel workbook.
jws-dvdy.21211.tbl5.xls34K Supplemental Table 5. Fold changes after cyclopamine treatment at tailbud stageEmbryos were treated with cyclopamine and microarray signals were determined at the tailbud stage and compared to wild type signals at the same stage. The fold change is as indicated for each microRNA with p-values as indicated.
jws-dvdy.21211.tbl6.xls4K Supplemental Table 6. Fold changes after cyclopamine treatment at 6 somite stageEmbryos were treated with cyclopamine and microarray signals were determined at the 6 somite stage and compared to wild type signals at the same stage. The fold change is as indicated for each microRNA. Since the effects of cyclopamine at later stages of development could be indirect, p-values were not determined for the 6 somite stage.
jws-dvdy.21211.tbl7.xls5K Supplemental Table 7. Fold changes after cyclopamine treatment at 18 somite stageEmbryos were treated with cyclopamine and microarray signals were determined at the 18 somite stage and compared to wild type signals at the same stage. The fold change is as indicated for each microRNA. Since the effects of cyclopamine at later stages of development could be indirect, p-values were not determined for the 18 somite stage.
jws-dvdy.21211.tbl8.xls5K Supplemental Table 8. Fold changes after cyclopamine treatment at 1 dpfEmbryos were treated with cyclopamine and microarray signals were determined at 1dpf and compared to wild type signals at the same stage. The fold change is as indicated for each microRNA. Since the effects of cyclopamine at later stages of development could be indirect, pvalues were not determined for 1 dpf.
jws-dvdy.21211.tbl9.xls35K Supplemental Table 9. Fold changes after DAPT treatment at tailbud stageEmbryos were treated with DAPT and microarray signals were determined at the tailbud stage and compared to wild type signals at the same stage. The fold change is as indicated for each microRNA with p-values as indicated.
jws-dvdy.21211.tbl10.xls4K Supplemental Table 10. Fold changes after DAPT treatment at 6 somite stageEmbryos were treated with DAPT and microarray signals were determined at the 6 somite stage and compared to wild type signals at the same stage. The fold change is as indicated for each microRNA. Since the effects of DAPT at later stages of development could be indirect, p-values were not determined for the 6 somite stage.
jws-dvdy.21211.tbl11.xls4K Supplemental Table 11. Fold changes after DAPT treatment at 18 somite stageEmbryos were treated with DAPT and microarray signals were determined at the 18 somite stage and compared to wild type signals at the same stage. The fold change is as indicated for each microRNA. Since the effects of DAPT at later stages of development could be indirect, p-values were not determined for the 18 somite stage.
jws-dvdy.21211.tbl12.xls5K Supplemental Table 12. Fold changes after DAPT treatment at 1 dpfEmbryos were treated with DAPT and microarray signals were determined at 1dpf and compared to wild type signals at the same stage. The fold change is as indicated for each microRNA. Since the effects of DAPT at later stages of development could be indirect, p-values were not determined for 1 dpf.

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