Comment[ArrayExpressAccession]	E-GEOD-65024			
MAGE-TAB Version	1.1			
Public Release Date	2015-01-16			
Investigation Title	Transcriptome analysis of Osbhlh148 mutant plants under controlled drought stress and well-watered conditions at vegetative stage			
Comment[Submitted Name]	Transcriptome analysis of Osbhlh148 mutant plants under controlled drought stress and well-watered conditions at vegetative stage			
Experiment Description	The members of bHLH transcription factor superfamily are known to play key role in plant development and abiotic stress response. Loss-of-function of OsbHLH148 gene resulted in increased sensitivity of rice plants to drought stress. To identify the targets of OsbHLH148 and dissect the drought stress response pathway regulated by it, we performed transcriptome profiling of Osbhlh148 mutant plants under drought stress as well as well-watered conditions by RNA-sequencing. OsbHLH148 loss-of-function rice plants (Oryza sativa ssp. japonica cv. Nipponbare) were exposed to controlled drought stress and well-watered conditions at the vegetative stage. Controlled drought (DR) stress was applied on 45 day old plants following gravimetric approach. The soil water content was brought down to 40% field capacity over a period of 3-4 days and plants were maintained at that level for 10 days by weighing the pots daily at a fixed time of the day and replenishing the water lost through evapotranspiration. Another set of plants were maintained at 100% FC as well-watered (WW) condition. Total RNA isolated from leaf tissue was used for RNA-sequencing. Two biological replicates per sample were sequenced. cDNA library was constructed using TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina). Sequencing was carried out on each library to generate 50 bp SE reads using Illumina High-Seq 2000 platform. The transcriptome reads were mapped to the rice reference genome sequence (MSU 7.0) with tophat1.3.1 using the program’s default parameters (http://tophat.cbcb.umd.edu). Mapped RNA-Seq reads were assembled into transcripts by Cufflinks (http://cufflinks.cbcb.umd.edu/) and differentially expressed genes were identified by using Cuffdiff.			
Term Source Name	ArrayExpress	EFO		
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl		
Person Last Name	Pereira	Venkategowda	Srivastava	Pereira
Person First Name	Andy	Ramegowda	Subodh	Andy
Person Mid Initials			K	
Person Email	apereira@uark.edu			
Person Affiliation	University of Arkansas,			
Person Phone	479-575-8435			
Person Address	Crop, Soil and Environmental Sciences (CSES), University of Arkansas,, 115 Plant Science Building,, Fayetteville, AR, USA			
Person Roles	submitter			
Protocol Name	P-GSE65024-4	P-GSE65024-1	P-GSE65024-3	P-GSE65024-2
Protocol Description	Trapnell et. al, Nature Protocols 7, 562–578 (2012) pipeline was used for analyses. The reads were Mapped with tophat v2.05 on MSU rice genome v7 information Transcript Assembly was done with cufflinksv2.0.2 Transcript merge with cuffmerge Cuffdiff used for Differencial expression Genome_build: MSU rice genome v7 information Supplementary_files_format_and_content: Cuffdiff output gene_exp.exp file was used for further analyses	Controlled drought stress was applied on 45 day old plants following gravimetric approach. The soil water content was brought down to 40% field capacity over a period of 3-4 days and plants were maintained at that level for 10 days by weighing the pots daily at a fixed time of the day and replenishing the water lost through evapotranspiration. Well-watered plants were maintained at 100% FC.	Total RNA was isolated using TRIZol reagent (Invitrogen) following the manufacturer's instructions. Total RNA was treated with RNase-free DNase I (Promega) and passed through RNeasy spin columns (Qiagen) to remove DNA and other contaminants. The concentration, quality and integrity of the RNA were analysed using the Agilent 2100 bioanalyzer (Bio-Rad) and NanoDrop™ ND-1000 (Thermo Scientific). Samples with RNA Quality Indicator (RQI) of > 8.0 were used for RNA-sequencing. Libraries were prepared using TruSeq Stranded Total RNA with Ribo-Zero Plant kit according to Illumina's instructions accompanying the Kit TruSeq Stranded Total RNA with Ribo-Zero	Rice plants were grown in the greenhouse conditions with 26 +/- 1 C day /22 +/- 1 C night temperature, 600 mmol/m2/s light intensity and 14 h light and 10 h dark cycles. All pots, filled with Redi-earth potting mix (Sun Gro Horticulture Distribution Inc., Bellevue, WA, USA), were placed in water-filled trays to simulate flooded condition and fertilized every week with 24-8-16 Miracle-Gro (Scotts Miracle-Gro Products, Inc., Marysville, OH, USA) until used for the experiment.
Protocol Type	normalization data transformation protocol	sample treatment protocol	nucleic acid library construction protocol	growth protocol
Experimental Factor Name	environmental stress	genotype		
Experimental Factor Type	environmental stress	genotype		
Comment[SecondaryAccession]	GSE65024			
Comment[GEOReleaseDate]	2015-01-16			
Comment[ArrayExpressSubmissionDate]	2015-01-15			
Comment[GEOLastUpdateDate]	2015-01-16			
Comment[AEExperimentType]	RNA-seq of coding RNA			
Comment[AdditionalFile:Data1]	GSE65024_WT-bhlh_gene_exp.diff			
Comment[SecondaryAccession]	SRP052309			
Comment[SequenceDataURI]	http://www.ebi.ac.uk/ena/data/view/SRR1761780-SRR1761787			
Comment[AEExperimentDisplayName]	Transcription profiling by high throughput sequencing of rice OsbHLH148 loss of function mutant under controlled drought stress and well-watered conditions at vegetative stage			
SDRF File	E-GEOD-65024.sdrf.txt