Chip was performed like described in Busch W, Miotk A, Ariel FD, Zhao Z, Forner J, Daum G, Suzaki T, Schuster C, Schultheiss SJ, Leibfried A, Haubeiss S, Ha N, Chan RL, Lohmann JU. (2010) Transcriptional control of a plant stem cell niche. Dev. Cell 18:849-61. except that DNA-free protein A agarose beads (Invitrogen) were used. Immunoprecipitation was performed using a rabbit polyclonal antibody against GFP (ab290, Abcam). DNA from the ChIP and mock experiments was amplified using a random-primer-based genome amplification method described in http://cat.ucsf.edu/pdfs/22_Round_A_B_C_protocol.pdf, with minor modifications.
Label
Cy3
Label protocol
After labelling with Cy3 and Cy5, respectively, following an amino-allyl-dye coupling protocol (http://camd.bio.indiana.edu/files/amino-allyl-protocol.pdf), DNA was cleaned up using the PCR purification kit (Qiagen) and 3 μg from the ChIP and from the mock samples were taken and mixed for hybridization to a custom long oligonucleotide (~60 bases) Arabidopsis promoter microarray.
Chip was performed like described in Busch W, Miotk A, Ariel FD, Zhao Z, Forner J, Daum G, Suzaki T, Schuster C, Schultheiss SJ, Leibfried A, Haubeiss S, Ha N, Chan RL, Lohmann JU. (2010) Transcriptional control of a plant stem cell niche. Dev. Cell 18:849-61. except that DNA-free protein A agarose beads (Invitrogen) were used. Immunoprecipitation was performed using a rabbit polyclonal antibody against GFP (ab290, Abcam). DNA from the ChIP and mock experiments was amplified using a random-primer-based genome amplification method described in http://cat.ucsf.edu/pdfs/22_Round_A_B_C_protocol.pdf, with minor modifications.
Label
Cy5
Label protocol
After labelling with Cy3 and Cy5, respectively, following an amino-allyl-dye coupling protocol (http://camd.bio.indiana.edu/files/amino-allyl-protocol.pdf), DNA was cleaned up using the PCR purification kit (Qiagen) and 3 μg from the ChIP and from the mock samples were taken and mixed for hybridization to a custom long oligonucleotide (~60 bases) Arabidopsis promoter microarray.
Hybridization protocol
Hybridization was performed according to the Agilent ChIP-chip protocol
Scan protocol
Images were obtained using an Agilent microarray scanner (model G2565BA) at a resolution of 5 μm. Signal extraction and initial data processing were done using the Agilent feature extraction software
Data processing
Signal ratio (FC) was used to obtain a Z-Score of the foldchange that was then used to conduct the analysis
