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.
Label
Cy5
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.
Genomic DNA was purified from nuclei of Drosophila S2 cells and fragmented by MNase digestion to an average size of 500-1000 bp.
Label
Cy3
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.
Hybridization 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.
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.
Data processing
Log base 2 (PM_532/PM_635) ratios were bi-weight mean centered
