ChIP experiment was performed on WT, emf1, emf2, transgenic FIE, and transgenic plants harboring the EMF1-3FLAG construct grown in short day growth conditions for 14 days. Plants were vacuum infiltrated in 1% formaldehyde solution for half an hour to cross-link the chromatin. Tissues were ground in liquid nitrogen, nuclei isolated, and chromatin extracted. Chromatin was sheared by sonication to 0.5-2 kb fragments. For immunoprecipitated chromatin (IP), monoclonal anti-FLAG mouse antibody (Sigma F1804) and polyclonal anti-H3K27me3 antibody (Upstate, rabbit IgG, 07-449) were added to fragmented chromatin to precipitate EMF1-bound and H3K27me3 modified chromatin, respectively. Cross-linking was reversed with 5M NaCl and DNA precipitated by 100% EtOH. For global gene expression studies, total RNA was extracted from 14-day-old WT and mutants using trizol and converted into cDNA. Genomic DNA from WT and cDNA from mutants and WT were labeled with CY3, and CY5, respectively, and combined to hybridize.
Label
Cy5
Label protocol
ChIP-chip was performed according to the NimbleGen protocol (Roche, www.nimblegen.com). IP and Input DNA were amplified using the Sigma Whole Genome Amplification kit and labeled with CY5 and CY3, respectively according to the NimbleGen protocol (www.nimblegen.com). Combined samples, which include 10ug of CY5-labeled IP and 10ug of CY3-labeled Input DNA, were hybridized with NimbleGen HD2 arrays.
ecotype: Columbia genotype/variation: WT sample type: input DNA (control)
Growth protocol
Short day growth conditions for 14 days
Extracted molecule
genomic DNA
Extraction protocol
ChIP experiment was performed on WT, emf1, emf2, transgenic FIE, and transgenic plants harboring the EMF1-3FLAG construct grown in short day growth conditions for 14 days. Plants were vacuum infiltrated in 1% formaldehyde solution for half an hour to cross-link the chromatin. Tissues were ground in liquid nitrogen, nuclei isolated, and chromatin extracted. Chromatin was sheared by sonication to 0.5-2 kb fragments. For immunoprecipitated chromatin (IP), monoclonal anti-FLAG mouse antibody (Sigma F1804) and polyclonal anti-H3K27me3 antibody (Upstate, rabbit IgG, 07-449) were added to fragmented chromatin to precipitate EMF1-bound and H3K27me3 modified chromatin, respectively. Cross-linking was reversed with 5M NaCl and DNA precipitated by 100% EtOH. For global gene expression studies, total RNA was extracted from 14-day-old WT and mutants using trizol and converted into cDNA. Genomic DNA from WT and cDNA from mutants and WT were labeled with CY3, and CY5, respectively, and combined to hybridize.
Label
Cy3
Label protocol
ChIP-chip was performed according to the NimbleGen protocol (Roche, www.nimblegen.com). IP and Input DNA were amplified using the Sigma Whole Genome Amplification kit and labeled with CY5 and CY3, respectively according to the NimbleGen protocol (www.nimblegen.com). Combined samples, which include 10ug of CY5-labeled IP and 10ug of CY3-labeled Input DNA, were hybridized with NimbleGen HD2 arrays.
Hybridization protocol
The hybridization and data extraction were performed at the Fred Hutchinson Cancer Research Center DNA array facility according to standard NimbleGen protocols (www.nimblegen.com).
