Web supplement to
"Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe"



Protocol for GeneChip microarray


The GeneChip microarray procedure was carried out according to the modified protocols described by Pierce et al. ( Nat Protoc. 2:2958-74 (2007), Nat Methods. 3:601-3 (2006)).

  1. Custom-made GeneChip from Affymetrix

  2. Preparation of genomic DNA

  3. PCR Amplification of tags

  4. Hybridization and chip scanning

  5. Recipes for hybridization solutions


Protocol for GeneChip microarray

The GeneChip microarray procedure was carried out according to the modified protocols described by Pierce et al. ( Nat Protoc. 2:2958-74 (2007), Nat Methods. 3:601-3 (2006)).


1. Custom-made GeneChip from Affymetrix
The custom-made GeneChip (48K) was designed according to the Affymetrix GeneChip guide (KRIBBSP2, Part No. 520506). Click here for full information.


2. Preparation of genomic DNA
Genomic DNA was prepared from frozen cell stocks (collected during the pooled growth experiments), using Zymo Research ZR-Fungal/ Bacterial DNA kit (catalog # D6005). For each sample, 10-20 OD600 corresponding to 2~4x108 cells/ml was used. ( http://www.zymoresearch.com/zrc/pdf/D6005i.pdf )


3. PCR Amplification of tags
To amplify and label the tags we used the following sets of primers and 0.2 μg of genomic DNA as template.


1) PCR primers for the labeling of UP-tag


2) PCR primers for the labeling of DOWN-tag
3) The PCR amplification step was carried out as follows:

  Concentration of stock solutions Concentration of working solutions Volume (μl)
PCR buffer 10X 1X 10
MgC12 50 mM 2.5 mM 5
dNTPs 10 mM 0.2 mM 2
Primer mix (UP or DN) 50 μM 1 μM 2
Taq polymerase 5 U/μl 5 U 1
Genomic DNA (~ 0.2 μg) 25
H2O 55
Total volume 100


4. Hybridization and chip scanning
Hybridization was carried out as follows using the Affymetrix Fluidics Station 450. The solutions and procedures in bold are described in section 5, Recipes for hybridization solutions.

  1. Fill the arrays (Affymetrix custom chips [KRIBBSP1, Tag 100K Array v1], Part No. 520429) with 140 μl 1X hybridization buffer.
  2. Pre-wet the array chips by incubating at 42°C for at least 10 min rotating at 20rpm in an Affymetrix GeneChip Hybridization Oven 640.
  3. While arrays are equilibrating, put 150.5 μl hybridization mix in a 0.5 ml micro tube.
  4. Boil each tube for 2 min and then put on ice for 2 min.
  5. Spin tubes briefly to bring the samples to the bottom of the tubes.
  6. Remove the hybridization buffer from each array and add samples.
  7. Place a Tough Tag over each gasket.
  8. Hybridize the array for 12-16 hrs at 42°C rotating at 20rpm.
  9. To prime the fluidics station: fill wash A, wash B, and water bottles, and run fluidics station protocol, PRIME_450.
  10. Prepare biotin labeling mix (do not freeze the mix).
  11. Aliquot 600 μ1 biotin staining mix per chip into tubes.
  12. Remove hybridization mix and fill chips with 140 μl wash A.
  13. Chips waiting to be washed should be wrapped in aluminum foil.
  14. Enter the sample name and the experiment name for each chip in the "Experiments" folder of the GCOS software.
  15. For staining and washes of chips, use the pombe TAG wash protocol in the "Fluidics" control window.
  16. Scan the arrays at an emission wavelength of 560 nm using an Affymetrix GeneArray Scanner.


5. Recipes for hybridization solutions

  1. 2x Hybridization buffer: 500 ml

    • (200 mM MES, 2 M NaCl , 40 mM EDTA, and 0.02% Tween20)
    • 83 ml of 12X MES stock
    • 177 ml of 5 M NaCl
    • 40 ml of 0.5 M EDTA
    • 1 ml of 10% Tween20
    • 199 ml of H2O
    • Store at 4°C (in the dark).

    1. 12X MES stock: 500 ml
      • (1.22 M MES and 0.89 M Na+):
      • 35 g of MES free acid monohydrate
      • 95 g of MES Sodium Salt
      • 400 ml of H2O
      • Mix and adjust volume to 500 ml.
        • The pH should be between 6.5 and 6.7.
        • Filter through a 0.2 mm filter.
        • Store at 4°C (in the dark).

  2. Hybridization mix: 150.5 μl
    • 75 μl of 2X hybridization buffer
    • 0.5 μl of text control oligonucleotide (20 fm/ml)
    • 12 μl of blocking oligo mix (12.5 or 37.5 pm/ml)
    • 3 μl of 50X Denhardt's solution (Sigma D-2532)
    • 60 μl of UP- and DOWN-tag PCR labeling solution (each 30 ml)
    • (See section 3, PCR amplification of tags in the Supplemental data 3.)

    1. Text control oligonucleotide:
    2. The biotinylated text control oligonucleotide hybridizes to the border of the microarray and shows the text "AFFX-KRIBBSP 1" at upper centre of the chip.

      5'-biotin-GTCGTCAAGATGCTACCGTTCAGGA-3'

      • Dissolve in H2O at 20 fm/μl.

    3. Blocking oligo mix:
    4. Blocking oligo mix consists of eight different oligos and is used to stop PCR- amplified tags hybridizing to each other through their common priming site and thus prevents probes of more than one tag hybridizing to the chip.
      In general, the amount of Up-tag PCR product is 2-3 times as much as that of Down-tag PCR product due to different priming efficiency. Therefore, three times higher amount of blocking oligo was used for shielding of Up-tag primers. The final concentrations of blocking oligo for shielding of Up- and Down- tags are 37.5 pmole/μl and 12.5 pmole/μl, respectively.

      Name Shielding for Sequence
      5U-Block F primer of Up tag (forward) 5'-CGCTCCCGCCTTACTTCGCATTTAAA-3'
      K5U-Block R primer of Up tag (forward) 5'-GGGGACGAGGCAAGCTAAGATATC-3'
      3U-Block R primer of Down tag (forward) 5'-TTGCGTTGCGTAGGGGGGATTTTAAA-3'
      K3U-Block F primer of Down tag (forward) 5'-CGCCATCCAGTGTCGAAAAGTATC-3'
      5U-Block (rev comp) F primer of Up tag (Reverse) 5'-TTTAAATGCGAAGTAAGGCGGGAGCG-3'
      K5U-Block (rev comp) R primer of Up tag (Reverse) 5'-GATATCTTAGCTTGCCTCGTCCCC-3'
      3U-Block (rev comp) R primer of Down tag (Reverse) 5'-TTTAAAATCCCCCCTACGCAACGCAA-3'
      K3U-Block (rev comp) F primer of Down tag (Reverse) 5'-GATACTTTTCGACACTGGATGGCG-3'

  3. Wash A (6X SSPE-Tween20): 1 L

    • 300 ml of 20x SSPE
    • 1 ml of 10% Tween20
    • 699 ml of H2O

  4. Wash B (3X SSPE-Tween20): 1 L

    • 150 ml of 20X SSPE
    • 1 ml of 10% Tween20
    • 849 ml of H2O

  5. Biotin labeling mix: 600 μl

    • 180.57 μl of 20X SSPE
    • 11.94 μl of 50X Denhardt's solution
    • 0.6 μl of 1% Tween20
    • 1.02 μl of 1 mg/ml streptavidin-phycoerythrin (Molecular Probes #S-866)
    • 405.87 μl of H2O

  6. Pombe TAG wash protocol: customised wash program of Fluidics P450:

  7. The built-in Affymetrix wash programme has been slightly modified to leave only one staining step. The details are as followings:

    1. Automatic washing using Affymetrix Fluidics Station 450
    2. Conditions No of Cycles Comment
      Wash A1 Recovery Mixes 0
      Wash A1 Temperature (°C)
      Number of Wash A1 Cycle 2
      Mixes per Wash A1 Cycle 4
      Wash B Recovery Mixes 0
      Wash B Temperature (°C) 42
      Number of Wash B Cycle 6
      Mixes per Wash B Cycle 4
      Stain Temperature (°C) 25
      First Stain Time (seconds) 0 (dummy step)
      Wash A2 Recovery Mixes 0 (dummy step)
      Wash A2 Temperature (°C) 25
      Number of Wash A2 Cycle 1
      Mixes per Wash A2 Cycle 2
      Second Stain Time (seconds) 600
      Third Stain Time (seconds) 0 (dummy step)
      Was A3 Recovery Mixes 0 (dummy step)
      Wash A3 Temperature (°C) 25
      Number of Wash A3 Cycles 6
      Mixes per Wash A3 Cycle 4
      Holding Temperature (°C) 25




Inquiries can be addressed to kwanghoe@kribb.re.kr.