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Sample GSM2645487 Query DataSets for GSM2645487
Status Public on Jul 12, 2017
Title FL1GR_4hD_FLAG3
Sample type genomic
Source name 8d sdlg, 4h DEX, anti-FLAG
Organism Arabidopsis thaliana
Characteristics genotype: Ws-0, 35S:FLAG-LEC1-GR
treatment: DEX
time: 4h
antibody: anti-FLAG
Treatment protocol 35S:FLAG-LEC1-GR seedlings were either (1) grown for 8 days on GM media (1X MS salts, 1% sucrose, 1X B5 vitamins, 0.8% Bactoagar, pH 5.7) and incubated for 4h in liquid GM media (same as solid, without Bactoagar) containing 30uM dexamethasone (DEX) or (2) germinated and grown on solid GM media containing 30 uM DEX for 8 days. Tissues were crosslinked under vaccuum for 10 minutes in Buffer A (0.4M sucrose, 10 mM Tris pH 8.0, 1mM EDTA pH 8.0) containing 1% formaldehyde, at room temperature, then quenched in 100 mM Glycine for 5 minutes. Crosslinked tissues were rinsed twice with deionized water, ground in liquid nitrogen and stored at -80C until use.
Growth protocol Plants were grown in a Percival cabinet under 16h day/8h night with fluorescent lamps at 20°C.
Extracted molecule genomic DNA
Extraction protocol Chromatin isolation and immunoprecipitation were performed as described in Gendrel AV, Lippman Z, Martienssen R, Colot V. Nat Methods. 2005;2:213–218, except that the isolated nuclei were resuspended in lysis buffer described in by Johnson et al. (2002) and sonicated to achieve chromatin fragments ranging from 100 to 500 bp. Nuclear extracts were either set aside for whole genome extracts (input control) or incubated overnight with 10ug of the anti-FLAG (Sigma F1804) antibody. Crosslinks were reversed and protein digested as in Dahl and Collas, Nat Protoc. 2008;3(6):1032-45, extracted with phenol:chloroform and precipitated using standard procedures.
Label biotin
Label protocol ChIP and Input control DNAs were prepared according to O'Geen, Nicolet, Bahnik, Green and Farnham, BioTechniques 41:577-580 (November 2006). 7.5 ug of DNA were fragmented to an average size of 100bp using RQ1 DNAseI (Promega), biotin-labelled using Affymetrix GeneChip WT Double Stranded DNA Terminal Labelling kit and following the manufacturer's instructions.
Hybridization protocol Samples were prepared and hybridized to GeneChip Arabidopsis Tiling 1.0R Array, according to Affymetrix specifications.
Scan protocol The tiling arrays were scanned using an Affymetrix GeneChip Scanner 3000 7G according to manufacturer's instruction.
Data processing Probe signal intensity data was processed and standardized using Model-based Analysis of Tiling-array software (MAT; BandWidth=200, MaxGap=150, MinProbe=8, Tvalue=0) to generate the .bar files containing the matscore for each probe on the array. The bar files were then used as input for CisGenome v1.2 to call significant regions (sample_peaks.bed) using the HMM algorithm and using a posterior probability (PP) > 0.99999 as threshold.
BAR files contain standardized probe intensity generated with Model-based Analysis of Tiling-array software (MAT; BandWidth=200, MaxGap=150, MinProbe=8, Tvalue=0). BED files contain the peaks called using CisGenome (v1.2) HMM algorithm, posterior probability > 0.99999. Chromosomal locations refer back to the TAIR8 genome assembly.
Submission date May 31, 2017
Last update date Jan 23, 2018
Contact name John Harada
Organization name University of California
Department Plant Biology
Lab Harada lab
Street address One Shields Avenue
City Davis
State/province CA
ZIP/Postal code 95616
Country USA
Platform ID GPL10977
Series (1)
GSE99529 Identification of LEC1 binding sites in Arabidopsis seedlings

Supplementary file Size Download File type/resource
GSM2645487_FL1GR_4hD_FLAG3.CEL.gz 39.6 Mb (ftp)(http) CEL 18.5 Mb (ftp)(http) BAR
Processed data provided as supplementary file
Processed data are available on Series record

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