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Protocols used in ChIP-on-chip experiments

Labeling of DNA and microarray hybridization:

Labeling of amplified DNA with fluorophores  200 ng of DNA from the previous step is mixed with 20 µl of 2.5X random primer solution (BioPrime kit, Invitrogen) and distilled H₂O in a final volume of 42.5 µl. The mixture is boiled at 95°C for 5 minutes and then cooled on ice for 5 minutes. Subsequently, 5 µl of 10X low dCTP mixture (1.2 mM each for dATP, dTTP and dGTP, and 0.6mM for dCTP), 1.5 µl of Cy5-dCTP (Amersham) or Cy3-dCTP (Amersham), and 40 u nits of Exo-Klenow DNA polymerase (BioPrime Kit, Invitrogen) are added to tube. The tube is incubated at 37°C for 1.5 hours. After the reaction, the labeled DNA is purified using the Qiaquick PCR clena-up kit (Qiagen).

Fabrication and hybridization of promoter DNA microarrays  An array (1.5K array) containing approximately 1500 human promoter fragments was initially designed based on NCBI human genome assembly Build 24 (July 3, 2001 freeze). 1200 gen es were selected from the collection of RefSeq entries based on three criteria: (1) their expression varied during the mammalian cell cycle; (2) the transcription start site (TSS) for each of these genes was annotated based on full-length mRNA s equences, and (3) their biological function was well documented. In addition, approximately 200 genes that do not exhibit cell cycle periodicity were also included. Oligonucleotide primers were designed to amplify fragments with endpoints at - 700 to +300 relative to the start site of these genes using PCR. After PCR, the amplified fragments were purified, verified by gel electrophoresis, and spotted onto GAPSII glass slides (Corning, Inc) using a Cartesian microarrayer (Genomic Solutions, Inc) . After UV crosslinking, the glass slides were stored in a vacuum desiccator until use.

In a new Eppendorf tube, 2.0 µg of Cy5-labeled ChIP-enriched DNA is mixed with 2.0 µg of Cy3-labeled genomic (control input) DNA and 36 µg of human Cot-1 DNA (Invitrogen). DNA is precipitated with 1/10 volume of 3M sodium acetate and 2 volumes of etha nol, and the DNA is dissolved in 22.4 µl of hybridization buffer 1. The mixture can be heated for 10 minutes at 37°C to facilitate resuspension of the DNA. 20 µl of hybridization buffer 2 is added to the mixture, and the tube is heated at 95°C for 5 min utes to denature DNA, then incubated at 42°C for 2 minutes. 4 µl of yeast tRNA (10 µg/µl; Sigma) and 3 µl of 2% BSA are then added to the mixture. While the labeled DNA is prepared, the microarrays are incubated in prehybridization solution (preheated to 42°C) for 40 minutes. They are then rinsed gently with distilled water and spon dry at 800rpm for 3 minutes. The resuspended probe is then spotted to the slide. A 25 mm x 60 mm cover slip is then gently placed on top of the sample, and the hybridi zation is carried out in a hybridization chamber (Corning) at 60°C overnight in a water bath.

After hybridization, the microarray slide is washed once with wash buffer 1 (2xSSC, 0.1% SDS) that has been preheated to 60°C for 5 minutes in a glass slide staining dish at room temperature. This is followed by a wash with buffer 2 (0.2XSSC, 0.1% SDS) for 10 minutes at room temperature and three times with buffer 3 (0.2XSSC), 1minute each, at room temperature. The slide is then dried by a brief spin at 1000 x g in a table-top centrifuge.