Roots were used of of 5-day-old seedlings expressing ARR1 genomic sequence under the control of its own promoter, fused to GFP (pARR1::ARR1:GFP plants)
Extracted molecule
genomic DNA
Extraction protocol
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.
Roots were used of of 5-day-old seedlings expressing ARR1 genomic sequence under the control of its own promoter, fused to GFP (pARR1::ARR1:GFP plants)
Extracted molecule
genomic DNA
Extraction protocol
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
For ChIP-chip analysis, to assess genome-wide binding the processed signal ratios of each experiment were randomly sampled 10,000 times and an empirical P-value was estimated for each ratio [like in 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]. The empirical P-values for each probe of the independent ChIP-chip experiments were multiplied.
