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Series GSE6996 Query DataSets for GSE6996
Status Public on Feb 21, 2007
Title Tiling microarray of Oryza sativa japonica at 46nt resolution
Organism Oryza sativa
Experiment type Expression profiling by genome tiling array
Summary This experiment was designed to identify transcribed regions of japonica subspecies of the rice genome. A series of high-density oligonucleotide tiling arrays that represent sense and antisense strands of the entire nonrepetitive sequence of all the 12 chromosomes were designed to measure genome-wide transcription. A total of 12253842 36mer oligonucleotide probes positioned every 46 nt on average were used for this purpose. The probes were synthesized via maskless photolithography at a feature density of approximately 389,000 probes per slide. The arrays were hybridized with fluorescence-labeled cDNA reverse-transcribed from equal amounts of four selected poly(A)+ RNA population (seedling root, seedling shoot, panicle, and suspension cultured cells).
Keywords: tiling array, genome-wide transcription
 
Overall design The arrays were hybridized with fluorescence-labeled cDNA reverse-transcribed from equal amounts of four selected poly(A)+ RNA population (seedling root, seedling shoot, panicle, and suspension cultured cells).

The four RNA population from seedling roots, seedling shoots, panicles, and suspension-cultured cells were pooled in equal amount and cDNAs were derived from the RNA mixture. Total RNA and mRNA were isolated using the RNeasy Plant Mini kit (Qiagen, Valencia, CA) and the Oligotex mRNA kit (Qiagen), respectively. First-strand cDNA was generated from 4 micro gram poly(A)+ RNA with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA).

The cDNAs were precipitated in ethanol:isopropanol (1:1 v/v) and resuspended in 0.1 M NaHCO3 to facilitate coupling of Alexa Fluor 555 NHS esters (Molecular Probes, Eugene, OR) to the reactive groups of the amino-allyl dUTPs and were purified with CyScribe GFX glass fiber spin columns (Amersham Bioscience, Piscataway, NJ).

Microarrays were hybridized with 4 ¦Ìg labeled cDNA in 300 micro litre hybridization buffer (50 mM MES, 0.5 M NaCl, 10 mM EDTA, and 0.005% (v/v) Tween-20) for 16 hours at 50 deg C in disposable adhesive chambers (Grace BioLabs, Bend, OR) in a hybridization oven with constant agitation. Hybridized arrays were washed on an orbital Sample in non-stringent buffer (6¡Á SSPE, 0.01% [v/v] Tween-20) for 10 minutes at room temperature, then in stringent buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween-20) for 30 minutes at 45 deg C. This was followed by a 5-minute wash in non-stringent buffer and a 2-minute wash in 0.1X SSC.

The arrays were dried with compressed nitrogen and scanned with an Axon 4000B laser scanner at 5 micro meter resolution.

The fluorescence intensity data were extracted with the NimbleScan software (NimbleGen Systems, Madison, WI).
 
Contributor(s) Li L, Wang X, Sasidharan R, Stolc V, Deng W, He H, Korbel J, Chen X, Tongprasit W, Ronald P, Chen R, Gerstein M, Deng X
Citation(s) 17372628
Submission date Feb 09, 2007
Last update date Oct 14, 2015
Contact name Rajkumar Sasidharan
E-mail(s) rajkumar.sasidharan@yale.edu
Organization name Yale University
Department MB & B
Lab Gerstein
Street address 266 Whitney Avenue
City New Haven
State/province CT
ZIP/Postal code 06520
Country USA
 
Platforms (32)
GPL4816 Tiling microarray for Oryza sativa japonica genome (version 2 pseudomolecules from TIGR) Chromosome 12
GPL4822 Tiling microarray OS16606 for Oryza sativa japonica genome (version 2 pseudomolecules from TIGR) Chromosome 2
GPL4823 Tiling microarray OS16618 for Oryza sativa japonica genome (version 2 pseudomolecules from TIGR) Chromosome 12
Samples (32)
GSM161100 Tiling microarray for Oryza sativa japonica genome (version 2 pseudomolecules from TIGR) Chromosome 12
GSM161142 Tiling microarray OS16606 for Oryza sativa japonica genome (version 2 pseudomolecules from TIGR) Chromosome 2
GSM161143 Tiling microarray OS16618 for Oryza sativa japonica genome (version 2 pseudomolecules from TIGR) Chromosome 12
Relations
BioProject PRJNA99273

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