• CamSysBiol
• Russell Lab
• DrosDel
• FlyMine
• FlyBase
• Proteomics

Affymetrix Processing of ChIP or DamID DNA to GeneChip Drosophila Tiling 2.0R Array

Outline

For the following protocol 7.5 µg DNA containing dUTP (see the corresponding DamID or ChIP protocols for Affymetrix microarrays for the sample preparation) are required for the hybridisation to GeneChip Drosophila Tiling 2.0R Array (49 Array Format).

Equipment and reagents

• GeneChip Drosophila Tiling 2.0R Array (Affymetrix; Cat. No. 901021)
• WT Double Stranded DNA Terminal Labeling Kit (Affymetrix; Cat. No. 900812)
• Control Oligo B2, 3 nM (Affymetrix; Cat. No. 900301)
• GeneChip Hybridization Wash and Stain Kit (Affymetrix; Cat. No. 900720)
• Affymetrix GeneChip Hybridization Oven 640
• Affymetrix GeneChip Fluidics Station 450
• Affymetrix GeneChip Scanner 3000 7G
• Hettich micro 20 centrifuge
• Grant QBT2 hot-block
• Dyad thermal cycler (PCR block)
• GenePix 4000B Microarray Scanner
• NimbleGen Hybridization System 4
• Gel electrophoresis equipment

Procedure

Fragmentation using the WT Double Stranded DNA Terminal Labeling Kit:

1. Prepare samples in PCR tubes as follows:
   • 7.5 µg Double-stranded DNA (purified PCR product)
   • 4.8 µL 10x cDNA Fragmentation Buffer
   • 1.5 µL 10U/µl UDG
   • 2.25 µL 100U/µl APE 1
2. Then make up to 48 µL using Nuclease-free water (supplied in kit)
3. Mix and spin
4. Incubate:
   • 37 °C for 2 hours
   • 93 °C for 2 minutes
   • Cool down to 4 °C
5. Run 2 µL of the reaction on a 1% agarose gel (fragment size should be 100-200 bp, otherwise the fragmentation time needs to be optimised)
6. Transfer 45 µL of the fragmented sample to a new PCR tube

Labelling:

7. Prepare labeling premix:
   • 12 µL 5x TdT Buffer
   • 2 µL TdT
   • 1 µL 5mM DNA Labeling Reagent
8. Add 15 µL of labeling premix to each sample
9. Mix and spin
10. Incubate:
    • 37 °C for 1 hour
    • 70 °C for 10 minutes
    • cool down to 4 °C
11. Switch on the GeneChip Hybridization Oven 640 and set the temperature to 45 °C to allow the temperature to stabilise

Hybridisation using the Hyb Module of the GeneChip Hybridization Wash and Stain Kit:

12. Equilibrate the GeneChip Drosophila Tiling 2.0R Array to room temperature
13. Prepare in 1.5 mL tubes:
    • 60 µL Fragmented and Labelled sample
    • 3.3 µL Control Oligo B2
    • 100 µL 2x Hybridisation Mix
    • 14 µL DMSO
14. Make up to 200 µL with Nuclease-free Water
15. Mix and spin
16. Heat at 99 °C for 5 minutes
17. Incubate to 45 °C for 5 minutes
18. Spin at 13,000 rpm for 1 minute
19. Label each array with the sample name
20. Insert a 250 µL pipette tip into the vent septa on the back of the array (see Affymetrix array schema below)
21. Inject 200 µL through the load septa while holding the array vertical
    Note: Use a 250 µL Filter tip for loading the sample. When using 200 µL Filter tips the sample can get absorbed into the filter when holding the pipette horizontal during the loading process!

Schema of Affymetrix array:

22. Remove the pipette tips and seal both septa with tough spots
23. Insert the array in the probe array tray and slide it into the GeneChip Hybridization Oven (make sure the carousel is balanced)
24. Position the small hooked end of the tray retainer over the upper rail of the carousel frame
25. Snap the lower end of the tray retainer over the lip of the probe array tray
26. Incubate at 45 °C, 60 rpm for 16 hours

Prime Fluidics Station (takes ~10 minutes):

27. Switch on the Fluidics Station and insert the wash line A into wash solution A and wash line B into wash solution B (light sensitive), make sure there is sufficient deionised water in the DI water bottle
28. Open AGCC Fluidics Control from the Affymetrix Launcher
29. On the screen tick the boxes of the Fluidics Modules to be used (one for each array)
30. Tick the List Maintenance Protocols Only option
31. Select PRIME_450 protocol
32. Click Copy to Selected Modules
33. Change to the Station 1 ID window
34. Click Run for each Module to be used
35. Follow the instructions on the Fluidics Station LCD window as the prime progresses:
    • When prompted raise the Needle Lever
    • Place three empty 1.5 mL tubes onto the Sample Holder
    • Lower the Needle Lever
36. Priming is finished when remove all vials message is displayed in the LCD window
37. Raise the Needle Lever and remove vials

Prepare Staining Reagents using the Stain Module of the GeneChip Hybridization Wash and Stain Kit:

38. For each array prepare three 1.5 mL tubes as follows:
    • Tube 1 - 600 µL Stain Cocktail 1 (light sensitive)
    • Tube 2 - 600 µL Stain Cocktail 2
    • Tube 3 - 800 µL Array Holding Buffer
39. Protect tubes from light until use

Prepare Arrays for staining:

40. Remove arrays from the oven after 16 hours
41. Insert a 250 µL pipette tip into the venting septa on the back of the array (see schema above)
42. Remove hybridisation cocktail from the array through the loading septa using a 250 µL pipette tip whilst holding the array vertical (the sample can be stored at -20 °C and re-hybridised in case of a problem with the array)
43. Load 250 µL Wash Buffer A using a 1000 µL pipette tip
44. Remove both pipette tips
45. Arrays which are not stained immediately should be kept at 4 °C in the dark

Create a New Project (skip when adding arrays to an existing project):

46. Open AGCC Portal from the Affymetrix Launcher
47. Go to ADMINISTRATION - Projects - Manage
48. Select the data root in the left panel
49. Add Subfolder name (e.g. your name or project name) and press Create
50. Click on the "new" Subfolder just created on the left panel
51. Add Project name and press Create

Register Samples:

52. Open AGCC Portal from the Affymetrix Launcher
53. Go to SAMPLES - Quick Register
54. Select number of sample files (ARR) to create from drop down list
55. Assign a project and Probe Array Type (DM_tiling2_MR_v01)
56. Add Sample File Name, Array Name and scan the barcode of the array
57. Press Next

Stain Arrays in Fluidics Station (takes ~1.5 hours):

58. In the Fluidics Control window press Refresh button
59. For each Module select Sample File Name from the drop down menu
60. In the Protocol selection tick the All box
61. Select Protocol: FS450_0001 (for Drosophila Tiling 2.0R Array)
62. Click Run for each Module to be used
63. Follow the instructions on the Fluidics Station LCD window as the prime progresses:
    • When fluidics LCD prompts lift the Cartridge Lever
    • Insert the array and lower the Cartridge Lever
    • When the fluidics LCD prompts raise the Needle Lever
    • Place the three Staining tubes onto their respective Sample Holder positions
    • Lower the Needle Lever
64. After ~1.5 hours when the fluidics LCD prompts:
    • Lift the Cartridge Lever and remove the array
    • Engage the Cartridge Lever
    • When the fluidics LCD prompts raise the Needle Lever
    • Place three empty 1.5 mL tubes onto the Sample Holder
    • Lower the Needle Lever
65. Run has finished when the LCR displays: Protocol done

Shutdown Fluidics Station (takes ~12 minutes):

66. Remove Wash Solution A and B and place all wash lines into the DI water bottle
67. In the Fluidics Control window select the Shutdown_450 protocol for all used Modules
68. Press Run for each Module
69. Follow the instructions on the Fluidics Station LCD window:
    • Raise the Needle Lever
    • Place three empty 1.5 mL tubes onto the Sample Holder
    • Lower the Needle Lever
70. After Shutdown protocol is completed switch off the Fluidics station

Scanning:

71. Inspect the array for air bubbles
72. In case of "larger" air bubbles:
    • Insert a 250 µL pipette tip into the venting septa on the back of the array (refer to schema above)
    • Remove solution through the loading septa using a 1000 µL pipette tip
    • Load 800 µL Array Holding Buffer
    • Remove both pipette tips
73. Seal both septa with tough spots
74. Let the scanner warm up for at least 10 minutes
75. Insert arrays in the carousel starting at position 1
76. Open the AGCC San Control from the Affymetrix Launcher
77. Select Edit - Options
    • Clear the enable Manual Mode check box
    • Tick box the Turn on Laser at Startup
    • OK
78. Press Start
79. After scan has finished remove arrays
80. Close Scan Control software and switch scanner off

Review the Grid Alignment using the AGCC Viewer:

81. Open the AGCC Viewer from the Affymetrix Launcher
82. Select the array image from the Review Window or select - File - Open File and browse for the *.DAT image
83. Press the Auto Scale button if the features are too bright
84. Navigate to the corners using the shortcut buttons on the left panel
85. Check the grid alignment at the corners and the middle of each array
86. In case the grid alignment has failed or is not optimal follow the help instructions of how to re-align the grids

Raw image files are named *.DAT. The *.CEL files contain the raw intensity values for each probe. Continue with the data normalisation and enrichment detection using any analysis tool of your choice.

B. Fischer (07-09-2010).