|
| Status |
Public on Mar 21, 2011 |
| Title |
meristem sample 2 (Me2) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
shoot apical meristem
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype: clavata3-9
tissue: meristem enriched tissue sample
chip antibody: H3K27me3
|
| Treatment protocol |
For tissue comparison meristematic tissue (Me) and young leaves (Le) were isolated by manual dissection of whole seedlings.
|
| Growth protocol |
All plants were grown for 9 weeks under short day conditions (8 h light/16 h darkness) on soil.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each tissue, 200 mg plant material was harvested on ice, crosslinked with 1% formaldehyde for 15 minutes under vacuum, frozen in liquid nitrogen and stored at -70° until chromatin extraction. A previously described protocol (Schubert D. et al., 2006 [EMBO J 25: 4638-4649]) was used for ChIP. Antibodies specific for H3K27me3 (#07-449) were retrieved from Millipore (Billerica, USA). Chromatin was sheared with a Bioruptor sonicator (10 pulses, 30 seconds each) (Diagenode, Liege, Belgium). Input and precipitated DNA was amplified with a whole genome amplification kit (WGA2, Sigma, St. Louis) as described in “PROT30” of The Epigenome NoE web-page (http://www.epigenome-noe.net/).
|
| Label |
Cy5
|
| Label protocol |
Probe labelling was performed by the Imagenes NimbleGen service (Berlin, Germany) as described in the “NimbleGen Array User’s Guide” protocol (http://www.nimblegen.com/products/lit/chip_userguide_v6p1.pdf) based on random priming with Cy3 or Cy5 nonamers.
|
| |
|
| Channel 2 |
| Source name |
shoot apical meristem
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype: clavata3-9
tissue: meristem enriched tissue sample
chip antibody: none
|
| Treatment protocol |
For tissue comparison meristematic tissue (Me) and young leaves (Le) were isolated by manual dissection of whole seedlings.
|
| Growth protocol |
All plants were grown for 9 weeks under short day conditions (8 h light/16 h darkness) on soil.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each tissue, 200 mg plant material was harvested on ice, crosslinked with 1% formaldehyde for 15 minutes under vacuum, frozen in liquid nitrogen and stored at -70° until chromatin extraction. A previously described protocol (Schubert D. et al., 2006 [EMBO J 25: 4638-4649]) was used for ChIP. Antibodies specific for H3K27me3 (#07-449) were retrieved from Millipore (Billerica, USA). Chromatin was sheared with a Bioruptor sonicator (10 pulses, 30 seconds each) (Diagenode, Liege, Belgium). Input and precipitated DNA was amplified with a whole genome amplification kit (WGA2, Sigma, St. Louis) as described in “PROT30” of The Epigenome NoE web-page (http://www.epigenome-noe.net/).
|
| Label |
Cy3
|
| Label protocol |
Probe labelling was performed by the Imagenes NimbleGen service (Berlin, Germany) as described in the “NimbleGen Array User’s Guide” protocol (http://www.nimblegen.com/products/lit/chip_userguide_v6p1.pdf) based on random priming with Cy3 or Cy5 nonamers.
|
| |
|
| |
| Hybridization protocol |
Array hybridisation was performed by the Imagenes NimbleGen service (Berlin, Germany) as described in the “NimbleGen Array User’s Guide” protocol (http://www.nimblegen.com/products/lit/chip_userguide_v6p1.pdf).
|
| Scan protocol |
Scanning was performed by the Imagenes NimbleGen service (Berlin, Germany) as described in the “NimbleGen Array User’s Guide” protocol (http://www.nimblegen.com/products/lit/chip_userguide_v6p1.pdf).
|
| Description |
tissue specific histone methylation
|
| Data processing |
For the processed data file gene specific methylation scores ranging from 0 to 100 were calculated as described below: The array hybridization data was received from Imagenes (Berlin, Germany) as intensity ratios of precipitate and input samples in the GFF-file format. In a first processing step M-Values [M=log2(IP/I) where IP is signal intensity from hybridized immunoprecipitated DNA and I is signal intensity from hybridized input DNA] were determined. The data was analyzed with SignalMap1.9 software (Nimblegen) and the Bioconductor R packages (Gentleman RC. et al., 2004, Genome Biol 5: R80) in an R programming environment. M values were used as input for the Bioconductor package Ringo. The Ringo clustering algorithm has been developed specifically for widely occurring enrichments including those found for histone modifications (Toedling J. et al., 2007, BMC Bioinformatics 8: 221). Prior to cluster detection Ringo applies a running median to smooth the datasets. A window size of 1400 bp was used for running median. The cluster algorithm was executed with a distance cut off of 700 bp and an intensity threshold defined by the 80th percentile. The identified methylation clusters were used to calculate gene specific methylation scores for each annotation [based on TAIR8 genome annotation (www.arabidopsis.org)] and tissue (Me or Le) and scaled so that the maximum equals 100. Further analyses and comparisons of loci-specific methylation scores were calculated with a regular spreadsheet program (Excel, Microsoft). A scale normalisation on the level of methylation scores was performed for the different tissues to account for differences in precipitation efficiency.
|
| |
|
| Submission date |
Sep 30, 2010 |
| Last update date |
Mar 21, 2011 |
| Contact name |
Daniel Schubert |
| E-mail(s) |
Daniel.Schubert@uni-duesseldorf.de
|
| Organization name |
Heinrich-Heine-University Duesseldorf
|
| Department |
Institute for Genetics
|
| Lab |
AG Schubert
|
| Street address |
Universitaetsstr. 1
|
| City |
Duesseldorf |
| ZIP/Postal code |
40225 |
| Country |
Germany |
| |
|
| Platform ID |
GPL3371 |
| Series (2) |
| GSE24474 |
H3K27 tri-methylation analysis of meristematic tissue (Me) and young leaves (Le) isolated by manual dissection of clv3-9 plants |
| GSE24507 |
Gene expression and H3K27 tri-methylation analysis of meristematic tissue (Me) and young leaves (Le) isolated by manual dissection of clv3-9 plants |
|
