title: 58885 H33FL_1/INPUT_2

status: Public on Mar 01 2007

description: Histones were identically tagged at their N-termini with biotin ligase recognition peptide (BLRP) (de Boer, E. et al. Proc Natl Acad Sci U S A 100, 7480-5 (2003), and Beckett, D. et al. Protein Sci 8, 921-9 (1999)) and expressed together with E. coli biotin ligase in Drosophila S2 cells. Nuclei were prepared as described (McKittrick, E., et al., Proc. Natl. Acad. Sci. USA 101, 1525-1530 (2004).), and treated with micrococcal nuclease (MNase) to digest chromatin to yield mostly mononucleosomes. Chromatin was extracted (input) using a modification of the procedure of Blower, M.D., et al. (Developmental Cell 2, 319-330 (2002)). Biotinylated histone-containing nucleosome particles (pull-down) were purified and DNA was extracted from both input and pull-down fraction for microarray analysis. DNA from input (3 ug) and pull-down (4 ug) was provided to NimbleGen Systems for differential labelling by random priming with Cy3- or Cy5 and hybridization to oligonucleotide arrays.  For each experiment, input and pull-down channel signal intensities and scaled log2(ratios) were provided by NimbleGen Systems.

hyb_protocol: Hybridization protocol provided and performed by NimbleGen (http://www.nimblegen.com/product s/chip/index.html). Set MAUI hybridization unit to 42 deg C and allow time for the temperatu re to stabilize. Based on the A_260 measurement, combine 13 micrograms each of the Test and Reference Samples into a single 1.5 ml microcentrifuge tube. Protect tube from light during handling to prevent photobleaching of the light-sensitive Cy dyes. Dry the combined contents  in a Speed-Vac on low heat. Resuspend the sample in 10.9microliter VWR water and vortex to completely dissolve the sample. Spin the tube down briefly to collect the contents in the bo ttom. Using the NimbleGen Array Reuse Kit, add the following to the resuspended sample: 19.5  microliter 2X Hybridization Buffer, 7.8 microliter Hybridization Component A, 0.4 microlite r Cy3 CPK6 50mer Oligo (50nM), 0.4 microliter Cy5 CPK6 50mer Oligo (100nM). CPK6 oligos are included in the hybridization as controls that hybridize to alignment features on the Nimble Gen arrays. They are required for proper extraction of array data from the scanned image. Mi x the tube briefly then spin down to collect the contents in the bottom and place at 95 deg C for 5 minutes. Immediately transfer the tube to the MAUI 42 deg C sample block and hold at  this temperature until you are are ready for sample loading. Place the MAUI Mixer SL Hybridization Chamber on the array using the provided assembly/disas sembly jig and carefully follow MAUI setup instructions. Use the braying tool to remove all air bubbles from the adhesive gasket around the outside of the hybridization chamber. Put th e array and hybridization chamber on the MAUI and allow 30 seconds for the chips to come up to temperature. Load the sample using the pipet supplied with the MAUI Station and following  manufacturer instructions. During the loading, a small amount (3-7 microliter) of the sampl e may flow out of the outlet port. Confirm that there are no bubbles in the chamber. If ther e are, very gently massage any bubbles to either of the ends, away from the center of the ar ray. Avoid applying too much pressure since this will force liquid out of the ports. Place t he loaded array into one of the four MAUI bays and let equilibrate for 30 seconds. Wipe off any sample leakage at the ports with a Kim-Wipe, and adhere MAUI stickers to both ports. Clo se the bay clamp and select mix mode B. Hold down the mix button to start mixing. Confirm th at the mixing is in progress before closing the cover. Hybridize the sample 16-20 hours. Prior to removing the array from the MAUI Hybridization Station, prepare the following solut ions. You will need two 250ml dishes of Wash I, and one each for Wash II and Wash III. One d ish for Wash I should be shallow and be wide enough to accomodate the array and mixer loaded  in the MAUI assembly/dissassembly jig. The lid from a 1000 microliter pipet tip box works w ell. Also, the buffer in the first Wash I dish should be heated to 42 deg C to help soften t he adhesive on the hybridization chamber; this will help prevent braking the array. Place th e remaining three wash solutions in 300 microliter Tissue-Tek slide staining dishes. Wash I:  225 ml VWR Water, 25ml 10X Wash Buffer I, 25microliter 1M DTT. Wash II: 225ml VWR Water, 25 ml 10X Wash Buffer II, 25microliter 1M DTT. Wash III: 225ml VWR Water, 25microliter 10X Wash  Buffer III, 25microliter 1M DTT. Remove chip from MAUI Hybridization Station, load it back into the MAUI assembly/disassembly jig, and immerse in the shallow 250ml Wash I at 42 deg C.  Peel the hybridization chamber off very slowly to prevent the slide from cracking. Do not l et the surface of the slide dry out at any point during washing. While the chip is submerged , carefully peel off the mixer. Gently agitate the chip in Wash I for 10-15 seconds. Transfe r the slide into a slide rack in the second dish of Wash I and incubate 2 minutes with agita tion. Transfer to Wash II and incubate 1 minute with agitation. Rock the disk to move the wa sh over the tops of the arrays. Transfer to Wash III and incubate for 15 sec with agitation.  Remove array and spin dry in array-drying unit for 1 minute. Store the dried array in a dar k desiccator.

scan_protocol: Sample scan protocol provided and performed by NimbleGen (http://www.nimblegen.com/products/ chip/index.html). Axon Scanner (model 4000B). Turn on the power of the scanner. The switch i s on the back right hand side. Launch GenePix Software 10 minutes before scanning to allow l asers to warm. Open the scanner door and open the side carriage. Place chip in carriage so t hat array is face down and barcode end is closest to you. Move the black lever on the left s ide of the carriage left until the array is lying flat in the carriage. Release the lever so  that the arry is pushed over gently to the right side of the carriage and held firmly. Clos e the carriage (you should hear a click) and slide the scanner door shut. Open the hardware settings. Select the following settings for scanning: Wavelength (532=Cy3, 635=Cy5). Set the  532 laser PMT Gain to 650 and the 635 laser PMT Gain to 750. Power=100%. Pixel Size=5m. Lin es to average=1. Focus position=0micrometer. Select Image/Ratio to view both channels simult aneously. Perform a preview scan by clicking on the fast-forward icon. Stop scan once the fu ll arry is in view by pressing the stop icon. Switch to zoom mode by clicking on the array i mage and pressing Z. Click and drag the mouse on the area in which you want to zoom. Center the image of the entire array area within the viewing window. Under Tools on the left side o f the screen, click the Scan Area icon. Click and drag to define a box that bounds the visib ile array area. The box dimensions should be stretched to create a box just slightly larger than the array area. It is critical to include all of the corner probes within the scan imag e. Scan chip by clicking the play icon. While scanning, set the zoom level to view the whole im age and adjust the brightness and contrast of the displayed image to eliminate visible satur ation. Based on the appearance of the array features, adjust the PMT setting as appropriate.  The features should be mostly yellow. If your features are mostly green, either decrease th e 532 PMT or raise the 635 PMT. If the features are mostly red or orange, either raise the 5 32 PMT or decrease the 635 PMT. Zoom the view to a region scanned under the most recent PMT settings and click on the Histogram tab at the top of the left side of the screen to check g lobal intensity of the features. On the top left side of the screen make sure you have the 5 32 and 635 wavelength boxes checked so both wavelength histograms are displayed. Make sure t he Log Axis box is checked. You want the red and green curves to be as close as possible to one another. If the red curve is above the green, lower the red PMT setting or raise the gre en PMT setting. You want the curve to have 1e-5 normalized counts at the 65,000 intensity le vel (saturation). This means that you have about 1% of the features saturated. The position of the two curves on top of each other is more important than hitting the 1% saturation mark .  The histogram only graphs the area of the image that is viewable in the screen on the ima ge tab. If the histogram is no longer changing then either the chip is done scanning or the area that you can see on the image screen has been scanned. After the PMT settings are prope rly adjusted, stop the current scan, do not save this image. Restart the scan under the new settings and wait until the scan has completed. Save the images. Make sure you save both the  532nm and 635nm images as separate single image tif files.

data_processing: Log base 2 (58885_635/58885_532) ratios were bi-weight mean centered

source_name: Chromatin affinity-purified DNA with tagged histone H3.3 region from Drosophila S2 cells

label_protocol:
Sample labeling protocol provided and performed by NimbleGen (http://www.nimblegen.com/produ
cts/chip/index.html). Prepare the following three solutions: 50X dNTP Mix, Random 9mer Buffe
r, 10X TE.  Dilute Cy5 dye-labeled 9mers to 1 O.D./42 microliter Random 9mer Buffer. Aliquot
 to 40 microliter individual reaction volumes in 0.2 milliliter thin-walled PCR tubes and st
ore at -20 degrees C. Assemble the following components in a 0.2 milliliter thin-walled PCR
tube: 1 microgram LM-PCR Sample and 40 microliter Cy5-9mer Primers. Heat denature samples in
 thermocycler at 98 degrees C for 10 minutes. Quick chill in ice water bath. Snap-chilling s
tep after denaturation is critical for efficiency labeling. Add the following: 10 microliter
s 50X dNTPmix, 8 microliters VWR deionized water, 2 microliters Kienow (50 U/microliter). Do
 not vortex after addition of Kienow. Mix well by pipetting 10 times. Spin down at low RPM t
o force contents to bottom of tube. Incubate at 37 degrees C for 2 hours in a thermocycler p
rotected from light. Stop the reaction by addition of EDTA: 10 microliter Stop Solution (0.5
 M EDTA). Transfer the reaction to a 1.5 milliliter tube. Precipitate the labeled samples by
 adding NaCl and isopropanol to each tube as indicated: 11.5 microliter 5M NaCl, 110 microli
ter Isopropanol. Vortex briefly after addition of 5M NaCl prior to addition of isopropanol.
Vortex. Incubate 10 minutes at room temperature in the dark. Centrifuge at maximum speed for
 10 minutes. Remove supernatant with pipette. Rinse pellet with 500 microliter 80% ice-cold
ethanol. Dislodge pellet from tube wall. Centrifuge at maximum speed for 2 minutes. Remove s
upernatant with pipette. Speed-Vac on low heat for 5 minutes until dry, protected from light
. Store labeled samples at -20 degrees C, protected from light. Rehydrate dried pellets in 2
5 microliter VWR deionized water. Spin tubes down briefly prior to opening. Vortex for 30 se
conds and quick spin to collect contents at bottom of tube. Continue to vortex or let site i
n the dark at room temp until the pellet is completely rehydrated, then vortex again and qui
ckly spin. Measure the A_260 in each sample. It is important to consume as little sample as
possible when performing this measurement. Typical yields range from 10 to 30 micrograms per
 reaction.

source_name: Input DNA

label_protocol:
Sample labeling protocol provided and performed by NimbleGen (http://www.nimblegen.com/produ
cts/chip/index.html). Prepare the following three solutions: 50X dNTP Mix, Random 9mer Buffe
r, 10X TE.  Dilute Cy3 dye-labeled 9mers to 1 O.D./42 microliter Random 9mer Buffer. Aliquot
 to 40 microliter individual reaction volumes in 0.2 milliliter thin-walled PCR tubes and st
ore at -20 degrees C. Assemble the following components in a 0.2 milliliter thin-walled PCR
tube: 1 microgram LM-PCR Sample and 40 microliter Cy3-9mer Primers. Heat denature samples in
 thermocycler at 98 degrees C for 10 minutes. Quick chill in ice water bath. Snap-chilling s
tep after denaturation is critical for efficiency labeling. Add the following: 10 microliter
s 50X dNTPmix, 8 microliters VWR deionized water, 2 microliters Kienow (50 U/microliter). Do
 not vortex after addition of Kienow. Mix well by pipetting 10 times. Spin down at low RPM t
o force contents to bottom of tube. Incubate at 37 degrees C for 2 hours in a thermocycler p
rotected from light. Stop the reaction by addition of EDTA: 10 microliter Stop Solution (0.5
 M EDTA). Transfer the reaction to a 1.5 milliliter tube. Precipitate the labeled samples by
 adding NaCl and isopropanol to each tube as indicated: 11.5 microliter 5M NaCl, 110 microli
ter Isopropanol. Vortex briefly after addition of 5M NaCl prior to addition of isopropanol.
Vortex. Incubate 10 minutes at room temperature in the dark. Centrifuge at maximum speed for
 10 minutes. Remove supernatant with pipette. Rinse pellet with 500 microliter 80% ice-cold
ethanol. Dislodge pellet from tube wall. Centrifuge at maximum speed for 2 minutes. Remove s
upernatant with pipette. Speed-Vac on low heat for 5 minutes until dry, protected from light
. Store labeled samples at -20 degrees C, protected from light. Rehydrate dried pellets in 2
5 microliter VWR deionized water. Spin tubes down briefly prior to opening. Vortex for 30 se
conds and quick spin to collect contents at bottom of tube. Continue to vortex or let site i
n the dark at room temp until the pellet is completely rehydrated, then vortex again and qui
ckly spin. Measure the A_260 in each sample. It is important to consume as little sample as
possible when performing this measurement. Typical yields range from 10 to 30 micrograms per
 reaction.

tag: Input DNA


title: Dp(2;2)Cam3/+ ovaries / Df(2L)JH/+ ovaries - 301

status: Public on Dec 15 2005

type: RNA

city: Bethesda

first: Brian

last: Oliver

state: MD

address: 50 South Drive

department: LBG

country: USA

institute: NIDDK, NIH

description: Whole adult Drosophila melanogaster Dp(2;2)Cam3/+  and Df(2L)JH/+ female 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).

molecule: total RNA

organism: Drosophila melanogaster

source_name: Dp(2;2)Cam3/+ ovaries

tag: Female Dp(2;2)Cam3/+ ovaries

source_name: Df(2L)JH/+ ovaries

tag: Female Df(2L)JH/+ ovaries


title: (X;AA) hstra ovarian tumor / (XX;AA) otu ovarian tumor - 238

description: Whole adult Drosophila melanogaster ct otu[1] v[24]/y w otu[17] and w/B[s]Y; P{ w+ hs-tra[83]}/+ flies were grown at 25C on PB medium (KD Medical, Columbia, MD) for 5 to 7 days post eclosion. Dissected ovarian tumors 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 iboGreen 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). 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).

source_name: (X;AA) hs-tra ovarian tumor

source_name: (XX;AA) otu[1]/otu[17] ovarian tumor


title: (XX;AA) Sxl ovarian tumors / (XX;AA) otu ovarian tumor - 219

description: Whole adult Drosophila melanogaster y Sxl[fs3]/y cm Sxl[7BO] and ct otu[1] v[24]/y w otu[17] flies were grown at 25C on PB medium (KD Medical, Columbia, MD) for 5 to 7 days post eclosion. Dissected ovarian tumors 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 iboGreen 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). 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).

source_name: (XX;AA) Sxl ovarian tumor

source_name: (XX;AA) otu ovarian tumor


title: dsx / y[1] w[67c] females - 36

description: Whole adult Drosophila melanogaster dsx[swe]/Df(3R)dsx[M+15] and y[1] w[67c] female 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).

tag: dsx[swe]/Df(3R)dsx[M+15] flies

source_name: y[1] w[67c] females

tag: y[1] w[67c] female flies


title: dsx / tra2B - 30

description: 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).

tag: (XX;AA) otu[1]/otu[17] ovarian tumor flies


title: MNase_1/Total_3

type: genomic

city: Seattle

first: Jorja

last: Henikoff

state: WA

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

description: Nuclei were prepared  from D. melanogaster S2 cells as described (McKittrick, E., et al., Proc. Natl. Acad. Sci. USA 101, 1525-1530 (2004).), and treated with micrococcal nuclease (MNase) to digest chromatin to yield mostly mononucleosomes. Chromatin was extracted using a modification of the procedure of Blower, M.D., et al. (Developmental Cell 2, 319-330 (2002)) and DNA was extracted (nucleosomal DNA= "Total") Genomic DNA was purified from nuclei with Proteinase K treatment followed by Phenol/Chloroform and Chloroform extractions and ethanol precipitation, then fragmented by MNase digestion to an average size of 500-1000 bp. Genomic and nucleosomal (total) DNA were provided to NimbleGen Systems for differential labelling by random priming with Cy3 or Cy5 and hybridization to oligonucleotide arrays.  For each experiment, Cy3 and Cy5 channel signal intensities and scaled log2(ratios) were provided by NimbleGen Systems.

hyb_protocol: Hybridization protocol provided and performed by NimbleGen (http://www.nimblegen .com/products/chip/index.html). Set MAUI hybridization unit to 42 deg C and allo w time for the temperature to stabilize. Based on the A_260 measurement, combine  13 micrograms each of the Test and Reference Samples into a single 1.5 ml micro centrifuge tube. Protect tube from light during handling to prevent photobleachi ng of the light-sensitive Cy dyes. Dry the combined contents in a Speed-Vac on l ow heat. Resuspend the sample in 10.9microliter VWR water and vortex to complete ly dissolve the sample. Spin the tube down briefly to collect the contents in th e bottom. Using the NimbleGen Array Reuse Kit, add the following to the resuspen ded sample: 19.5 microliter 2X Hybridization Buffer, 7.8 microliter Hybridizatio n Component A, 0.4 microliter Cy3 CPK6 50mer Oligo (50nM), 0.4 microliter Cy5 CP K6 50mer Oligo (100nM). CPK6 oligos are included in the hybridization as control s that hybridize to alignment features on the NimbleGen arrays. They are require d for proper extraction of array data from the scanned image. Mix the tube brief ly then spin down to collect the contents in the bottom and place at 95 deg C fo r 5 minutes. Immediately transfer the tube to the MAUI 42 deg C sample block and  hold at this temperature until you are are ready for sample loading. Place the MAUI Mixer SL Hybridization Chamber on the array using the provided as sembly/disassembly jig and carefully follow MAUI setup instructions. Use the bra ying tool to remove all air bubbles from the adhesive gasket around the outside of the hybridization chamber. Put the array and hybridization chamber on the MAU I and allow 30 seconds for the chips to come up to temperature. Load the sample using the pipet supplied with the MAUI Station and following manufacturer instru ctions. During the loading, a small amount (3-7 microliter) of the sample may fl ow out of the outlet port. Confirm that there are no bubbles in the chamber. If there are, very gently massage any bubbles to either of the ends, away from the center of the array. Avoid applying too much pressure since this will force liqu id out of the ports. Place the loaded array into one of the four MAUI bays and l et equilibrate for 30 seconds. Wipe off any sample leakage at the ports with a K im-Wipe, and adhere MAUI stickers to both ports. Close the bay clamp and select mix mode B. Hold down the mix button to start mixing. Confirm that the mixing is  in progress before closing the cover. Hybridize the sample 16-20 hours. Prior to removing the array from the MAUI Hybridization Station, prepare the fol lowing solutions. You will need two 250ml dishes of Wash I, and one each for Was h II and Wash III. One dish for Wash I should be shallow and be wide enough to a ccomodate the array and mixer loaded in the MAUI assembly/dissassembly jig. The lid from a 1000 microliter pipet tip box works well. Also, the buffer in the fir st Wash I dish should be heated to 42 deg C to help soften the adhesive on the h ybridization chamber; this will help prevent braking the array. Place the remain ing three wash solutions in 300 microliter Tissue-Tek slide staining dishes. Was h I: 225 ml VWR Water, 25ml 10X Wash Buffer I, 25microliter 1M DTT. Wash II: 225 ml VWR Water, 25ml 10X Wash Buffer II, 25microliter 1M DTT. Wash III: 225ml VWR Water, 25microliter 10X Wash Buffer III, 25microliter 1M DTT. Remove chip from M AUI Hybridization Station, load it back into the MAUI assembly/disassembly jig, and immerse in the shallow 250ml Wash I at 42 deg C. Peel the hybridization cham ber off very slowly to prevent the slide from cracking. Do not let the surface o f the slide dry out at any point during washing. While the chip is submerged, ca refully peel off the mixer. Gently agitate the chip in Wash I for 10-15 seconds.  Transfer the slide into a slide rack in the second dish of Wash I and incubate 2 minutes with agitation. Transfer to Wash II and incubate 1 minute with agitati on. Rock the disk to move the wash over the tops of the arrays. Transfer to Wash  III and incubate for 15 sec with agitation. Remove array and spin dry in array- drying unit for 1 minute. Store the dried array in a dark desiccator.

scan_protocol: Sample scan protocol provided and performed by NimbleGen (http://www.nimblegen.c om/products/chip/index.html). Axon Scanner (model 4000B). Turn on the power of t he scanner. The switch is on the back right hand side. Launch GenePix Software 1 0 minutes before scanning to allow lasers to warm. Open the scanner door and ope n the side carriage. Place chip in carriage so that array is face down and barco de end is closest to you. Move the black lever on the left side of the carriage left until the array is lying flat in the carriage. Release the lever so that th e arry is pushed over gently to the right side of the carriage and held firmly. Close the carriage (you should hear a click) and slide the scanner door shut. Op en the hardware settings. Select the following settings for scanning: Wavelength  (532=Cy3, 635=Cy5). Set the 532 laser PMT Gain to 650 and the 635 laser PMT Gai n to 750. Power=100%. Pixel Size=5m. Lines to average=1. Focus position=0microme ter. Select Image/Ratio to view both channels simultaneously. Perform a preview scan by clicking on the fast-forward icon. Stop scan once the full arry is in vi ew by pressing the stop icon. Switch to zoom mode by clicking on the array image  and pressing Z. Click and drag the mouse on the area in which you want to zoom.  Center the image of the entire array area within the viewing window. Under Tool s on the left side of the screen, click the Scan Area icon. Click and drag to de fine a box that bounds the visibile array area. The box dimensions should be str etched to create a box just slightly larger than the array area. It is critical to include all of the corner probes within the scan image. Scan chip by clicking the play icon. While scanning, set the zoom level to view the whole image and adjust the brightness and contrast of the displayed image to  eliminate visible saturation. Based on the appearance of the array features, ad just the PMT setting as appropriate. The features should be mostly yellow. If yo ur features are mostly green, either decrease the 532 PMT or raise the 635 PMT. If the features are mostly red or orange, either raise the 532 PMT or decrease t he 635 PMT. Zoom the view to a region scanned under the most recent PMT settings  and click on the Histogram tab at the top of the left side of the screen to che ck global intensity of the features. On the top left side of the screen make sur e you have the 532 and 635 wavelength boxes checked so both wavelength histogram s are displayed. Make sure the Log Axis box is checked. You want the red and gre en curves to be as close as possible to one another. If the red curve is above t he green, lower the red PMT setting or raise the green PMT setting. You want the  curve to have 1e-5 normalized counts at the 65,000 intensity level (saturation) . This means that you have about 1% of the features saturated. The position of t he two curves on top of each other is more important than hitting the 1% saturat ion mark.  The histogram only graphs the area of the image that is viewable in t he screen on the image tab. If the histogram is no longer changing then either t he chip is done scanning or the area that you can see on the image screen has be en scanned. After the PMT settings are properly adjusted, stop the current scan,  do not save this image. Restart the scan under the new settings and wait until the scan has completed. Save the images. Make sure you save both the 532nm and 6 35nm images as separate single image tif files.

data_processing: Log base 2 (PM_532/PM_635) ratios were bi-weight mean centered

molecule: genomic DNA

label_protocol:
Sample labeling protocol provided and performed by NimbleGen (http://www.nimbleg
en.com/products/chip/index.html). Prepare the following three solutions: 50X dNT
P Mix, Random 9mer Buffer, 10X TE.  Dilute Cy3 dye-labeled 9mers to 1 O.D./42 mi
croliter Random 9mer Buffer. Aliquot to 40 microliter individual reaction volume
s in 0.2 milliliter thin-walled PCR tubes and store at -20 degrees C. Assemble t
he following components in a 0.2 milliliter thin-walled PCR tube: 1 microgram LM
-PCR Sample and 40 microliter Cy3-9mer Primers. Heat denature samples in thermoc
ycler at 98 degrees C for 10 minutes. Quick chill in ice water bath. Snap-chilli
ng step after denaturation is critical for efficiency labeling. Add the followin
g: 10 microliters 50X dNTPmix, 8 microliters VWR deionized water, 2 microliters
Kienow (50 U/microliter). Do not vortex after addition of Kienow. Mix well by pi
petting 10 times. Spin down at low RPM to force contents to bottom of tube. Incu
bate at 37 degrees C for 2 hours in a thermocycler protected from light. Stop th
e reaction by addition of EDTA: 10 microliter Stop Solution (0.5 M EDTA). Transf
er the reaction to a 1.5 milliliter tube. Precipitate the labeled samples by add
ing NaCl and isopropanol to each tube as indicated: 11.5 microliter 5M NaCl, 110
 microliter Isopropanol. Vortex briefly after addition of 5M NaCl prior to addit
ion of isopropanol. Vortex. Incubate 10 minutes at room temperature in the dark.
 Centrifuge at maximum speed for 10 minutes. Remove supernatant with pipette. Ri
nse pellet with 500 microliter 80% ice-cold ethanol. Dislodge pellet from tube w
all. Centrifuge at maximum speed for 2 minutes. Remove supernatant with pipette.
 Speed-Vac on low heat for 5 minutes until dry, protected from light. Store labe
led samples at -20 degrees C, protected from light. Rehydrate dried pellets in 2
5 microliter VWR deionized water. Spin tubes down briefly prior to opening. Vort
ex for 30 seconds and quick spin to collect contents at bottom of tube. Continue
 to vortex or let site in the dark at room temp until the pellet is completely r
ehydrated, then vortex again and quickly spin. Measure the A_260 in each sample.
 It is important to consume as little sample as possible when performing this me
asurement. Typical yields range from 10 to 30 micrograms per reaction.

extract_protocol: Nuclei were prepared from Drosophila S2 cells as described (McKittrick, E., et al., Proc. Natl. Acad. Sci. USA 101, 1525-1530 (2004).), and treated with micrococcal nuclease (MNase) to digest chromatin to yield mostly mononucleosomes. Chromatin was extracted (total) in extraction buffer (PBS supplemented with 0.213M NaCl (final 0.35M), 2mM EDTA, 0.5mM PMSF, and 0.1%Triton) using a modification of the procedure of Blower, M.D., et al. (Developmental Cell 2, 319-330 (2002)). Total DNA was purified for microarray analysis and provided to NimbleGen Systems for differential labelling by random priming with Cy3- or Cy5 and hybridization to oligonucleotide arrays.

tag: Nucleosomal DNA from Drosophila S2 cells

extract_protocol: Genomic DNA was purified from nuclei of Drosophila S2 cells and fragmented by MNase digestion to an average size of 500-1000 bp.

tag: genomic DNA from Drosophila S2 cells


title: (X;AA) hs-tra ovarian tumor / (XX;AA) WT ovary - 210

description: Whole adult Drosophila melanogaster w/B[s]Y; P{w+ hs-tra[83]}/+ and y[1] w[67c] female flies were grown at 25C on PB medium (KD Medical, Columbia, MD) for 5 to 7 days post eclosion. Dissected ovarian tumors 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 iboGreen 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). 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).


title: (XX;AA) otu ovarian tumor vs (X;AA) hs-tra ovarian tumor - 237

source_name: (XX;AA) otu[1]/ otu[17] ovarian tumors

source_name: (X;AA) hs-tra ovarian tumors


title: 58906 H3_1/Input_1

data_processing: Log base 2 (58906_635/58906_532) ratios were bi-weight mean centered

source_name: Chromatin affinity-purified DNA with tagged histone H3 from Drosophila S2 cells

label_protocol:
Sample labeling protocol provided and performed by NimbleGen (http://www.nimbleg
en.com/products/chip/index.html). Prepare the following three solutions: 50X dNT
P Mix, Random 9mer Buffer, 10X TE.  Dilute Cy5 dye-labeled 9mers to 1 O.D./42 mi
croliter Random 9mer Buffer. Aliquot to 40 microliter individual reaction volume
s in 0.2 milliliter thin-walled PCR tubes and store at -20 degrees C. Assemble t
he following components in a 0.2 milliliter thin-walled PCR tube: 1 microgram LM
-PCR Sample and 40 microliter Cy5-9mer Primers. Heat denature samples in thermoc
ycler at 98 degrees C for 10 minutes. Quick chill in ice water bath. Snap-chilli
ng step after denaturation is critical for efficiency labeling. Add the followin
g: 10 microliters 50X dNTPmix, 8 microliters VWR deionized water, 2 microliters
Kienow (50 U/microliter). Do not vortex after addition of Kienow. Mix well by pi
petting 10 times. Spin down at low RPM to force contents to bottom of tube. Incu
bate at 37 degrees C for 2 hours in a thermocycler protected from light. Stop th
e reaction by addition of EDTA: 10 microliter Stop Solution (0.5 M EDTA). Transf
er the reaction to a 1.5 milliliter tube. Precipitate the labeled samples by add
ing NaCl and isopropanol to each tube as indicated: 11.5 microliter 5M NaCl, 110
 microliter Isopropanol. Vortex briefly after addition of 5M NaCl prior to addit
ion of isopropanol. Vortex. Incubate 10 minutes at room temperature in the dark.
 Centrifuge at maximum speed for 10 minutes. Remove supernatant with pipette. Ri
nse pellet with 500 microliter 80% ice-cold ethanol. Dislodge pellet from tube w
all. Centrifuge at maximum speed for 2 minutes. Remove supernatant with pipette.
 Speed-Vac on low heat for 5 minutes until dry, protected from light. Store labe
led samples at -20 degrees C, protected from light. Rehydrate dried pellets in 2
5 microliter VWR deionized water. Spin tubes down briefly prior to opening. Vort
ex for 30 seconds and quick spin to collect contents at bottom of tube. Continue
 to vortex or let site in the dark at room temp until the pellet is completely r
ehydrated, then vortex again and quickly spin. Measure the A_260 in each sample.
 It is important to consume as little sample as possible when performing this me
asurement. Typical yields range from 10 to 30 micrograms per reaction.

extract_protocol: Histones were identically tagged at their N-termini with biotin ligase recognition peptide (BLRP) (de Boer, E. et al. Proc Natl Acad Sci U S A 100, 7480-5 (2003), and Beckett, D. et al. Protein Sci 8, 921-9 (1999)) and expressed together with E. coli biotin ligase in Drosophila S2 cells. Nuclei were prepared as described (McKittrick, E., et al., Proc. Natl. Acad. Sci. USA 101, 1525-1530 (2004).), and treated with micrococcal nuclease (MNase) to digest chromatin to yield mostly mononucleosomes. Chromatin was extracted (input) using a modification of the procedure of Blower, M.D., et al. (Developmental Cell 2, 319-330 (2002)). Biotinylated histone-containing nucleosome particles (pull-down) were purified and DNA was extracted from both input and pull-down fraction for microarray analysis. DNA from input (3 ug) and pull-down (4 ug) was provided to NimbleGen Systems for differential labelling by random priming with Cy3- or Cy5 and hybridization to oligonucleotide arrays.  For each experiment, input and pull-down channel signal intensities and scaled log2(ratios) were provided by NimbleGen Systems.


title: Dp(2;2)Cam3/+ male vs Df(2L)JH/+ male - 231

description: Whole adult Drosophila melanogaster y[1] w[67c]/Y; Dp(2;2)Cam3/+ male adult flies and y[1] w[67c]/Y; Df(2L)JH/+ male adult flies were grown at 25C on PB medium (KD Medical, Columbia, MD) for 5 to 7 days post eclosion. Whole flies 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). 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).

hyb_protocol: see description

scan_protocol: see description

molecule: polyA RNA

source_name: Dp(2;2)Cam3/+ male adult flies

label_protocol: see description

extract_protocol: see description

source_name: Df(2L)JH/+ male adult flies


title: Sonication_1/Total_10

description: Nuclei were prepared  from D. melanogaster S2 cells as described (McKittrick, E., et al., Proc. Natl. Acad. Sci. USA 101, 1525-1530 (2004).), and treated with micrococcal nuclease (MNase) to digest chromatin to yield mostly mononucleosomes. Chromatin was extracted using a modification of the procedure of Blower, M.D., et al. (Developmental Cell 2, 319-330 (2002)) and DNA was extracted (nucleosomal DNA= "Total") Genomic DNA was purified from nuclei with Proteinase K treatment followed by Phenol/Chloroform and Chloroform extractions and ethanol precipitation, then fragmented by sonication to an average size of 500-1000 bp. Genomic and nucleosomal (total) DNA were provided to NimbleGen Systems for differential labelling by random priming with Cy3 or Cy5 and hybridization to oligonucleotide arrays.  For each experiment, Cy3 and Cy5 channel signal intensities and scaled log2(ratios) were provided by NimbleGen Systems.

extract_protocol: Genomic DNA was purified from nuclei of Drosophila S2 cells and fragmented by sonication to an average size of ~500 bp.


title: Df(2L)JH/+ male vs Dp(2;2)Cam3/+ male - 230

description: Drosophila melanogaster y[1] w[67c] /Y; Df(2L)JH/Y male adult flies and y[1] w[67c] /+;Dp(2;2)Cam3/+ male adult flies flies were grown at 25C on PB medium (KD Medical, Columbia, MD) for 5 to 7 days post eclosion. Whole flies 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). 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).


title: (XX;AA) Sxl ovarian tumor vs (X;AA) hs-tra ovarian tumor - 225

description: Whole adult Drosophila melanogaster (XX;AA) y Sxl[fs3] / y cm Sxl[7BO] and w/B[s]Y; P{w[+] hs-tra[83]}/+ flies were grown at 25C on PB medium (KD Medical, Columbia, MD) for 5 to 7 days post eclosion. Dissected ovarian tumors 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 iboGreen 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). 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).

source_name: (XX;AA) y Sxl[fs3] / y cm Sxl[7BO] ovarian tumors

source_name: (X;AA) w/B[s]Y; P{w[+] hs-tra[83]}/+ ovarian tumors


title: (XX;AA) Sxl ovarian tumor vs (X;AA) hs-tra ovarian tumor - 232

description: Whole adult Drosophila melanogaster (XX;AA) y Sxl[fs3] / y cm Sxl[7BO] and (X;AA) y[1] w[67c]/Y; Df(3L)st[j7] Ki roe p[p] P{hs-tra}/+ flies were grown at 25C on PB medium (KD Medical, Columbia, MD) for 5 to 7 days post eclosion. Dissected ovarian tumors 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 iboGreen 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). 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).

source_name: (X;AA) y[1] w[67c]/Y; Df(3L)st[j7] Ki roe p[p] P{hs-tra}/+ ovarian tumors


title: Dp(2;2)Cam3/+ vs Df(2L)JH/+ female - 222

description: Drosophila melanogaster y[1] w[67c] /+; Dp(2;2)Cam3/+  and y[1] w[67c] /+; Df(2L)JH/+ female adult flies were grown at 25C on PB medium (KD Medical, Columbia, MD) for 5 to 7 days post eclosion. Whole flies 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 iboGreen 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). 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).

source_name: Dp(2;2)Cam3/+ female adult

source_name: Df(2L)JH/+ female adult


title: (X;AA) hstra ovarian tumor / (XX;AA) otu ovarian tumor - 217

description: Whole adult Drosophila melanogaster ct otu[1] v[24]/y w otu[17] and y[1] w[67]/Y; Df(3L) st[j7] Ki roe p[p] P{hs-tra}/+ flies were grown at 25C on PB medium (KD Medical, Columbia, MD) for 5 to 7 days post eclosion. Dissected ovarian tumors 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 iboGreen 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). 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).


title: (XX;AA) otu ovarian tumor vs (X;AA) hstra ovarian tumor- 212

description: Whole adult Drosophila melanogaster ct otu[1] v[24]/y w otu[17] and y[1] w[67c]/Y; Df(3L) st[j7] Ki roe p[p] P{hs-tra}/+  flies were grown at 25C on PB medium (KD Medical, Columbia, MD) for 5 to 7 days post eclosion. Dissected ovarian tumors 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 iboGreen 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). 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).


title: Df(2L)J-H/+ vs Dp(2;2)Cam3/+ female - 221

description: Drosophila melanogaster y[1] w[67c] /+; Df(2L)JH/+ female adult flies and y[1] w[67c] /+; Dp(2;2)Cam3/+ female adult flies were grown at 25C on PB medium (KD Medical, Columbia, MD) for 5 to 7 days post eclosion. Whole flies 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 iboGreen 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). 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).

source_name: Df(2L)J-H/+ female adult


title: (X;AA) B[s]Y; hs-tra carcass vs (XX;AA) Female Carcass - 34

description: Drosophila melanogaster (X;AA) B[s]Y; Df(3L)st[j7] Ki roe p[p] P{hs-tra}/+ sex transformed females and y[1] w[67c] female flies were grown at 25C on GIF medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion. Gonadectomized carcasses 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 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. 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. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 50 mM TrisHCl 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). Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA) at 535 nm. Signal intensities were initially stored and analyzed using GEMTools software (Incyte Genomics, Palo Alto CA). Background corrections were done by calculating the average Cy3/Cy5 signal to produce a balanced coefficient (Yue et al., 2001) Further corrections for non-uniform conditions during array printing and/or hybridization were performed using Qualifier software (Novation Bioscience, Palo Alto 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).

source_name: (X;AA) B[s]Y; hs-tra carcass female

source_name: (XX;AA) y[1] w[67c] Female Carcass


title: (XX;AA) dsx male vs (X:AA) tudor male - 35

description: Drosophila melanogaster dsx[swe]/Df(3R)dsx[M+15] and tud[1] bw[1] sp[1] male progeny of tud[1] bw[1] sp[1] mothers were grown at 25C on GIF medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion. Whole flies 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 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. 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. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 50 mM TrisHCl 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). Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA) at 535 nm. Signal intensities were initially stored and analyzed using GEMTools software (Incyte Genomics, Palo Alto CA). Background corrections were done by calculating the average Cy3/Cy5 signal to produce a balanced coefficient (Yue et al., 2001) Further corrections for non-uniform conditions during array printing and/or hybridization were performed using Qualifier software (Novation Bioscience, Palo Alto 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).

source_name: (XX;AA) dsx[swe]/Df(3R)dsx[M+15] sex transformed male

source_name: (X:AA) tud[1] bw[1] sp[1] male


title: (X;AA) B[s]Y; hs-tra carcass vs (X;AA) Male Carcass - 29

description: Drosophila melanogaster (X;AA) w/B[s]Y; P{w[+] hs-tra[83]}/+ sex transformed females and y[1] w[67c] male flies were grown at 25C on GIF medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion. Gonadectomized carcasses 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 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. 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. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 50 mM TrisHCl 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). Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA) at 535 nm. Signal intensities were initially stored and analyzed using GEMTools software (Incyte Genomics, Palo Alto CA). Background corrections were done by calculating the average Cy3/Cy5 signal to produce a balanced coefficient (Yue et al., 2001) Further corrections for non-uniform conditions during array printing and/or hybridization were performed using Qualifier software (Novation Bioscience, Palo Alto 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).

source_name: (X;AA) y[1] w[67c] Male Carcass


title: (X;AA) hs-tra carcass vs (XX;AA) pi[[2]]/ y w - 28

description: Drosophila melanogaster (X;AA) B[s]Y; P{w[+] hs-tra[83]}/+ sex transformed females and pi[[2]]/ y w female flies that show hybrid dysgenesis in the germline were grown at 25C on GIF medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion. The whole fly in pi[[2]]/ y w and the gonadectomized carcass of hs-tra flies 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 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. 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. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 50 mM TrisHCl 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). Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA) at 535 nm. Signal intensities were initially stored and analyzed using GEMTools software (Incyte Genomics, Palo Alto CA). Background corrections were done by calculating the average Cy3/Cy5 signal to produce a balanced coefficient (Yue et al., 2001) Further corrections for non-uniform conditions during array printing and/or hybridization were performed using Qualifier software (Novation Bioscience, Palo Alto 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).


title: Wang4570_JA261

status: Public on Feb 23 2005

city: San Diego

first: Tim

last: Jatkoe

state: CA

institute: Veridex

description: RNA was extracted from a fresh-frozen primary breast tumor. Target cRNA was prepared and hybridized using standard Affymetrix procedures.  Image was globally scaled to a target intensity of 600.

organism: Homo sapiens

source_name: Breast

tag: bone relapses (1=yes, 0=no)


title: Wang4831_JA_625


title: Wang4834_JA_630


title: Wang689_JA-67


title: Wang4558_JA247


title: Wang4896_JA_855@2


title: Wang4816_JA_284


title: Wang675_JA-46


title: Wang4572_JA264


title: Wang4058_JA-86


title: Wang4891_JA_848@2


title: Wang4059_JA-88


title: Wang4824_JA_612


title: Wang4852_JA_754


title: Wang4039_JA-137


title: Wang4821_JA_606


title: Wang4032_JA-125


title: Wang4888_JA_845


title: Wang4848_JA_738


title: Wang4628_JA791@2


title: Wang4830_JA_621


title: RV82.07_HYP_day10_Slide56

status: Public on Feb 01 2006

city: Leiden

first: Henk

last: Buermans

state: ZH

address: Albinusdreef 2

department: Human Genetics

country: Netherlands

institute: Leiden University Medical Center

description: day 10 post 30mg monocrotaline/kg bw injection

data_processing: standardized ratio between standardized sample intrensity vs. Reference pool intensity

organism: Rattus norvegicus

extract_protocol: Polytron - TriZol

tag: Strain

value: Wistar, Sex:male

source_name: Reference pool

tag: pooled total RNA from interventricular septum tissue


title: CON - LV21

status: Public on Aug 18 2004

description: CON - 14days - LV

source_name: CON - LV21


title: CON - R22

description: CON - 14days - RV

source_name: CON - R22


title: CON - LV22

description: CON - 14days - LV

source_name: CON - LV22


title: HYP - LV05

description: HYP - 14days - LV

source_name: HYP - LV05


title: CON - R17

description: CON - 14days - RV

source_name: CON - R17


title: CHF - LV09

description: CHF - 14days - LV

source_name: CHF - LV09


title: CON - R18

description: CON - 14days - RV

source_name: CON - R18


title: CHF - R10

description: CHF - 14days - RV

source_name: CHF - R10


title: CON - R19

description: CON - 14days - RV

source_name: CON - R19


title: HYP - R08

description: HYP - 14days - RV

source_name: HYP - R08


title: CON - R21

description: CON - 14days - RV

source_name: CON - R21


title: CHF - R11

description: CHF - 14days - RV

source_name: CHF - R11


title: CON - R20

description: CON - 14days - RV

source_name: CON - R20


title: HYP - LV03

description: HYP - 14days - LV

source_name: HYP - LV03


title: CHF - R15

description: CHF - 14days - RV

source_name: CHF - R15


title: HYP - R05

description: HYP - 14days - RV

source_name: HYP - R05


title: CHF - R09

description: CHF - 14days - RV

source_name: CHF - R09

source_name: Reference pool


title: CHF - LV15

description: CHF - 14days - LV

source_name: CHF - LV15


title: CHF - R14

description: CHF - 14days - RV

source_name: CHF - R14


title: CHF - R12

description: CHF - 14days - RV

source_name: CHF - R12


title: HYP - R04

description: HYP - 14days - RV

source_name: HYP - R04


title: HYP - R06

description: HYP - 14days - RV

source_name: HYP - R06


title: HYP - R03

description: HYP - 14days - RV

source_name: HYP - R03


title: HYP - R01

description: HYP - 14days - RV

source_name: HYP - R01


title: H3_#2/Total_2

status: Public on Sep 11 2005

description: Histones were identically tagged at their N-termini with biotin ligase recognition peptide (BLRP) (de Boer, E. et al. Proc Natl Acad Sci U S A 100, 7480-5 (2003), and Beckett, D. et al. Protein Sci 8, 921-9 (1999)) and expressed together with E. coli biotin ligase in Drosophila S2 cells. Nuclei were prepared as described (McKittrick, E., et al., Proc. Natl. Acad. Sci. USA 101, 1525-1530 (2004).), and treated with micrococcal nuclease (MNase) to digest chromatin to yield mostly mononucleosomes. Chromatin was extracted (input) using a modification of the procedure of Blower, M.D., et al. (Developmental Cell 2, 319-330 (2002)). Biotinylated histone-containing nucleosome particles (pull-down) were purified and DNA was extracted from both input and pull-down fraction for microarray analysis. DNA from input (3 ug) and pull-down (4 ug) was provided to NimbleGen Systems for differential labelling by random priming with Cy3- or Cy5 and hybridization to oligonucleotide arrays.  For each experiment, input and pull-down channel signal intensities and scaled log2(ratios) were provided by NimbleGen Systems.

data_processing: Log base 2 (PM_635/PM_532) ratios were bi-weight mean centered

source_name: Chromatin affinity-purified DNA with tagged histone H3 from Drosophila S2 cells

source_name: Input DNA


title: H33FL_1-2/H3_2-2

source_name: Chromatin affinity-purified DNA with tagged histone H3.3 region from Drosophila S2 cells

tag: Chromatin affinity-purified DNA with tagged histone H3.3 from Drosophila S2 cells

source_name: Chromatin affinity-purified DNA with tagged histone H3 region from Drosophila S2 cells

tag: Chromatin affinity-purifed DNA with tagged histone H3 region from Drosophila S2 cells


title: tudor male vs ovaries-19b

status: Public on Jun 28 2004

description: Germlineless adult Drosophila melanogaster tud[1] bw[1]sp[1] progeny of tud[1] bw[1]sp[1] mothers and adult y[1]w[67c] flies were grown at 25C on GIF medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion. Whole flies (tudor males) and dissected y[1]w[67c] 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 (Probe 1) or Cy5 (Probe 2). To synthesize probes, mRNA 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 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. 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. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 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). Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA) at 535 nm. Signal intensities were initially stored and analyzed using GEMTools software (Incyte Genomics, Palo Alto CA). Background corrections were done by calculating the average Cy3/Cy5 signal to produce a balanced coefficient (Yue et al., 2001) Further corrections for non-uniform conditions during array printing and/or hybridization were performed using Qualifier software (Novation Bioscience, Palo Alto CA).

source_name: Adult tud[1] bw[1]sp[1] Male

source_name: Adult y[1]w[67c] Ovaries


title: tudor Female vs testes-20b

description: Germlineless adult Drosophila melanogaster tud[1] bw[1]sp[1] progeny of tud[1] bw[1]sp[1] mothers and adult y[1]w[67c] flies were grown at 25C on GIF medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion. Whole flies (tudor females) and dissected y[1]w[67c] testes 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 (Probe 1) or Cy5 (Probe 2). To synthesize probes, mRNA 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 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. 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. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 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). Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA) at 535 nm. Signal intensities were initially stored and analyzed using GEMTools software (Incyte Genomics, Palo Alto CA). Background corrections were done by calculating the average Cy3/Cy5 signal to produce a balanced coefficient (Yue et al., 2001) Further corrections for non-uniform conditions during array printing and/or hybridization were performed using Qualifier software (Novation Bioscience, Palo Alto CA).

source_name: Adult tud[1] bw[1]sp[1] Female

source_name: Adult y[1]w[67c] Testes


title: yw Female vs tudor Female-25

description: Germlineless adult Drosophila melanogaster tud[1] bw[1]sp[1] progeny of tud[1] bw[1]sp[1] mothers and adult y[1]w[67c] flies were grown at 25C on GIF medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion. Whole flies 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 (Probe 1) or Cy5 (Probe 2). To synthesize probes, mRNA 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 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. 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. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 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). Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA) at 535 nm. Signal intensities were initially stored and analyzed using GEMTools software (Incyte Genomics, Palo Alto CA). Background corrections were done by calculating the average Cy3/Cy5 signal to produce a balanced coefficient (Yue et al., 2001) Further corrections for non-uniform conditions during array printing and/or hybridization were performed using Qualifier software (Novation Bioscience, Palo Alto CA).

source_name: Adult y[1]w[1] Female


title: H33dN_2/TOTAL_3

source_name: Chromatin affinity-purified DNA with tagged histone H3.3 core region from Drosophila S2 cells


title: XY;tra2 vs XX;tra2-16a

description: Adult Drosophila melanogaster w[67c]/w[67c];tra2[B]/TRIX and sibling w[67c]/BsY;tra2[B]/TRIX flies were grown at 25C on GIF medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion. Whole flies 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 (Probe 1) or Cy5 (Probe 2). To synthesize probes, mRNA 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 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. 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. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 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). Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA) at 535 nm. Signal intensities were initially stored and analyzed using GEMTools software (Incyte Genomics, Palo Alto CA). Background corrections were done by calculating the average Cy3/Cy5 signal to produce a balanced coefficient (Yue et al., 2001) Further corrections for non-uniform conditions during array printing and/or hybridization were performed using Qualifier software (Novation Bioscience, Palo Alto CA).

source_name: Adult w[67c]/BsY;tra2[B]/TRIX

source_name: Adult w[67c]/w[67c];tra2[B]/TRIX


title: tudor male vs tudor female-17a

description: Germlineless adult Drosophila melanogaster tud[1] bw[1]sp[1] progeny of tud[1] bw[1]sp[1] mothers were grown at 25C on GIF medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion. Whole flies 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 (Probe 1) or Cy5 (Probe 2). To synthesize probes, mRNA 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 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. 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. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 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). Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA) at 535 nm. Signal intensities were initially stored and analyzed using GEMTools software (Incyte Genomics, Palo Alto CA). Background corrections were done by calculating the average Cy3/Cy5 signal to produce a balanced coefficient (Yue et al., 2001) Further corrections for non-uniform conditions during array printing and/or hybridization were performed using Qualifier software (Novation Bioscience, Palo Alto CA).


title: tudor Female vs testes-20a


title: tudor male vs testes-18a

description: Germlineless adult Drosophila melanogaster tud[1] bw[1]sp[1] progeny of tud[1] bw[1]sp[1] mothers and adult y[1]w[67c] flies were grown at 25C on GIF medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion. Whole flies (tudor males) and dissected y[1]w[67c] testes 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 (Probe 1) or Cy5 (Probe 2). To synthesize probes, mRNA 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 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. 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. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 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). Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA) at 535 nm. Signal intensities were initially stored and analyzed using GEMTools software (Incyte Genomics, Palo Alto CA). Background corrections were done by calculating the average Cy3/Cy5 signal to produce a balanced coefficient (Yue et al., 2001) Further corrections for non-uniform conditions during array printing and/or hybridization were performed using Qualifier software (Novation Bioscience, Palo Alto CA).


title: H33FL_2/TOTAL_9

source_name: Chromatin affinity-purified DNA with tagged histone 3.3 region from Drosophila S2 cells


title: tudor male vs tudor female-17b


title: Male vs tudor Male-23

source_name: Adult y[1]w[1] Male


title: tudor Female vs ovaries-21b

description: Germlineless adult Drosophila melanogaster tud[1] bw[1]sp[1] progeny of tud[1] bw[1]sp[1] mothers and adult y[1]w[67c] flies were grown at 25C on GIF medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion. Whole flies (tudor females) and dissected y[1]w[67c] 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 (Probe 1) or Cy5 (Probe 2). To synthesize probes, mRNA 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 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. 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. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 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). Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA) at 535 nm. Signal intensities were initially stored and analyzed using GEMTools software (Incyte Genomics, Palo Alto CA). Background corrections were done by calculating the average Cy3/Cy5 signal to produce a balanced coefficient (Yue et al., 2001) Further corrections for non-uniform conditions during array printing and/or hybridization were performed using Qualifier software (Novation Bioscience, Palo Alto CA).


title: XY;tra2 vs XX;tra2-16b


title: tudor male vs ovaries-19a


title: tudor male vs yw Male-22

source_name: Adult y[1]w[67c] Male


title: tudor Female vs ovaries-21a


title: XX;tra2 vs XY;tra2-15b

description: Adult Drosophila melanogaster w[67c]/w[67c];tra2[B]/TRIX and sibling Adult w[67c]/BsY;tra2[B]/TRIX flies were grown at 25C on GIF medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion. Whole flies 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 (Probe 1) or Cy5 (Probe 2). To synthesize probes, mRNA 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 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. 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. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 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). Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA) at 535 nm. Signal intensities were initially stored and analyzed using GEMTools software (Incyte Genomics, Palo Alto CA). Background corrections were done by calculating the average Cy3/Cy5 signal to produce a balanced coefficient (Yue et al., 2001) Further corrections for non-uniform conditions during array printing and/or hybridization were performed using Qualifier software (Novation Bioscience, Palo Alto CA).


title: XX;tra2 vs XY;tra2-15a


title: tudor male vs testes-18b


title: tudor Female vs yw Female-24

source_name: Adult y[1]w[67c] Female