The whole above ground tissue was collected from 12 day old de-etiolated seedlings that were grown in the dark at 30oC in 1:1 mix of autoclaved field soil and MetroMix. For embryo, immature ear (approximately 5 – 7 cm in length), tassel, and endosperm collection, plants were grown to maturity in the field of the University of Minnesota Saint Paul Agricultural Research station during the summer of 2011. Endosperm and embryo were harvested from multiple B73 and Mo17 ears 14 days after the self-pollination. The seeds for each replicate came from a unique, single source (ear). Each biological replicate was planted at a different time and represents the pooled above ground tissue from eight to ten plants. Three replicate tissue collections were performed for all B73 and Mo17 tissues. For each replicate / tissue / genotype combination, 1 g of plant material was harvested on ice, rinsed with water, and crosslinked with 1% formaldehyde for 10 minutes under vacuum infiltration. Cross-linking was quenched by adding Glycine solution to a final concentration of 0.125M under vacuum infiltration for 5 minutes. Treated tissue was frozen in liquid nitrogen and stored at -800C until chromatin extraction.
Label
Cy3
Label protocol
Chromatin extractions were performed using EpiQuik™ Plant ChIP Kit (Epigentek, Brooklyn, USA) according to manufacturer’s recommendations. Extracted chromatin was sheared in 600 μl of the EpiQuick buffer CP3F with 5 10-second pulses on a sonicator. To test and optimize sonication conditions, cross-linking was reversed in a sample of sheared chromatin and the resulting products were analyzed on agarose gel. Sonication conditions were optimized to yield predominantly 200 – 500 bp DNA samples. Chromatin immunoprecipitations, reverse cross-linking, and DNA cleanup were performed using EpiQuik™ Plant ChIP Kit (Epigentek) according to manufacturer’s recommendations. Antibodies specific for H3K27me3 (#07-449) were purchased from Millipore (Billerica, USA). For each genotype, antibody, and replicate, 50 – 100 ng of input and immunoprecipitated (IP) DNA was amplified with a whole genome amplification kit (WGA2, Sigma, St. Louis, USA). The amplification of no antibody control (negative control) was always 5 – 10 fold less efficient confirming specificity of immunoprecipitation. For each amplified IP and input sample, 3 ug of amplified DNA were labeled using the Dual-Color Labeling Kit (Roche NimbleGen, Cat # 05223547001) according to the array manufacturer’s protocol (Roche NimbleGen Methylation User- Guide v7.0). Each IP sample was labeled with Cy5 and each input/control sonicated DNA was labeled with Cy3.
genotype: mez3_m4 in B73 background tissue: seedling ChIP: input
Extracted molecule
genomic DNA
Extraction protocol
The whole above ground tissue was collected from 12 day old de-etiolated seedlings that were grown in the dark at 30oC in 1:1 mix of autoclaved field soil and MetroMix. For embryo, immature ear (approximately 5 – 7 cm in length), tassel, and endosperm collection, plants were grown to maturity in the field of the University of Minnesota Saint Paul Agricultural Research station during the summer of 2011. Endosperm and embryo were harvested from multiple B73 and Mo17 ears 14 days after the self-pollination. The seeds for each replicate came from a unique, single source (ear). Each biological replicate was planted at a different time and represents the pooled above ground tissue from eight to ten plants. Three replicate tissue collections were performed for all B73 and Mo17 tissues. For each replicate / tissue / genotype combination, 1 g of plant material was harvested on ice, rinsed with water, and crosslinked with 1% formaldehyde for 10 minutes under vacuum infiltration. Cross-linking was quenched by adding Glycine solution to a final concentration of 0.125M under vacuum infiltration for 5 minutes. Treated tissue was frozen in liquid nitrogen and stored at -800C until chromatin extraction.
Label
Cy5
Label protocol
Chromatin extractions were performed using EpiQuik™ Plant ChIP Kit (Epigentek, Brooklyn, USA) according to manufacturer’s recommendations. Extracted chromatin was sheared in 600 μl of the EpiQuick buffer CP3F with 5 10-second pulses on a sonicator. To test and optimize sonication conditions, cross-linking was reversed in a sample of sheared chromatin and the resulting products were analyzed on agarose gel. Sonication conditions were optimized to yield predominantly 200 – 500 bp DNA samples. Chromatin immunoprecipitations, reverse cross-linking, and DNA cleanup were performed using EpiQuik™ Plant ChIP Kit (Epigentek) according to manufacturer’s recommendations. Antibodies specific for H3K27me3 (#07-449) were purchased from Millipore (Billerica, USA). For each genotype, antibody, and replicate, 50 – 100 ng of input and immunoprecipitated (IP) DNA was amplified with a whole genome amplification kit (WGA2, Sigma, St. Louis, USA). The amplification of no antibody control (negative control) was always 5 – 10 fold less efficient confirming specificity of immunoprecipitation. For each amplified IP and input sample, 3 ug of amplified DNA were labeled using the Dual-Color Labeling Kit (Roche NimbleGen, Cat # 05223547001) according to the array manufacturer’s protocol (Roche NimbleGen Methylation User- Guide v7.0). Each IP sample was labeled with Cy5 and each input/control sonicated DNA was labeled with Cy3.
Hybridization protocol
Samples were hybridized to the custom 1.4 or 2.1 M probe array for 16–20 hrs at 42C.
Scan protocol
Slides were washed and scanned according to NimbleGen’s protocol for the MS200 microarray scanner. Images were aligned and quantified using NimbleScan software (Roche NimbleGen) producing raw data reports for each probe on the array.
Data processing
XYS files exported from NimbleScan were imported into the Bioconductor statistical environment (http://bioconductor.org/). Sample-dependent H3K27me3 enrichments were estimated for each probe by fitting a fixed linear model accounting for array, dye, and sample effects to the data using the limma package (Wettenhall and Smyth, 2004). Moderated t-statistics and the log-odds score for differential MeDIP enrichment was computed by empirical Bayes shrinkage of the standard errors with the False Discovery Rate controlled to 0.05. Results were visualized using Integrated Genomics Viewer (Robinson et al., 2011)
