MAGE-TAB Version	1.1
Investigation Title	RNA-seq Transcriptional Profiling of Herbaspirillum seropedicae Colonizing Bread Wheat (Triticum aestivum) Roots.

Experimental Design	growth condition design	replicate design	validation by reverse transcription PCR design
Experimental Design Term Source REF	EFO	EFO	EFO
Experimental Design Term Accession Number	EFO_0001759	EFO_0001776	OBI_0001162

Experimental Factor Name	growth condition
Experimental Factor Type	growth condition
Experimental Factor Term Source REF	EFO
Experimental Factor Term Accession Number	EFO_0000523

Person Last Name	Pankievicz	Balsanelli
Person First Name	Vânia	Eduardo
Person Mid Initials	CS	
Person Email	vaniacarla@gmail.com	balsanelli@ufpr.br
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Person Affiliation	UFPR	UFPR
Person Address		
Person Roles		submitter
Person Roles Term Source REF	
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Quality Control Type
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Replicate Type
Replicate Term Source REF
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Normalization Type
Normalization Term Source REF
Normalization Term Accession Number

Date of Experiment	2013-06-06
Public Release Date	2016-07-03

PubMed ID
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Experiment Description	H. seropedicae is a diazotrophic and endophytic bacterium that associates with economically important grasses promoting plant growth and increasing productivity. To identify genes related to bacterial ability to colonize and promote plant growth wheat seedlings growing hydroponically in Hoagland’s medium were inoculated with H. seropedicae the bacteria and incubated for 3 days. mRNA from the bacteria present in the root surface and in the plant medium were purified, depleted from rRNA and used for RNA-seq profiling. RT-qPCR analyses were conducted to confirm regulation of selected genes. Comparison of RNA profile of bacteria attached to the root and planktonic revealed an extensive metabolic adaptation to the epiphytic life style.

Protocol Name	P-MTAB-45177	P-MTAB-45176	P-MTAB-45173	P-MTAB-45175	P-MTAB-45174
Protocol Type	high throughput sequence alignment protocol	nucleic acid sequencing protocol	growth protocol	nucleic acid library construction protocol	nucleic acid extraction protocol
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO
Protocol Term Accession Number	EFO_0004917	EFO_0004170	EFO_0003789	EFO_0004184	EFO_0002944
Protocol Description	'The sequences obtained were mapped on the H. seropedicae genome using the software CLC Genomics Workbench (v. 6.5.1). The following parameters were used: reads were trimmed to minimum of 40 pb, 90% alignment to the reference sequence and 80% similarity required for inclusion as a mapped read, number of hits equal to 1, number of additional bases down and upstream of the CDS equal to 50 pb. Expressed genes were those that had more than 3 times of coverage. The differencial gene expression analyses was performed using DESeq (DESeq using R-package from Bioconductir’s project) and were considered regulated those with a fold- change greater than or equal to two and significance level in the Baggerley''s test higher than 95%, FDR ≤ 0.05. RPKM values (Mortazavi et al. 2008) were calculated using CLC Genomics Workbench (v. 6.5.1).'	Sequenced in next generation sequencing platform SOLiD 4 (Life Technologies) using the ToP Sequencing Kit (1 x50bp).	H. seropedicae growth conditions The H. seropedicae strain SmR1 was grown routinely at 30° C with shaking at 120 rotations per minute (rpm) in NFbHP-malate medium (Klassen et al. 1997) with 20 mM of NH4Cl added (NFbHPN-Malate). Streptomycin was added as needed at the concentrations of 80 μg/mL. The bacterial strains for plant inoculation were pre- cultured overnight in 5 mL of NFbHPN-malate medium containing NH4Cl and antibiotics as needed. The overnight culture was used to inoculate 10mL NFbHPN-malate medium, which was grown as previously described to an OD600 = 1.0. The bacterial cells were collected by quick centrifugation (14.000 rpm for 20 seconds at room temperature) to ensure that the bacteria remained viable. Cell pellets were re-suspended in the same volume of Hoagland’s medium (Hoagland 1950) to a cell density of 107 cells/mL. A volume of 250 μL of this bacterial suspension was added to glass tube containing 25 mL of Hoagland’s medium and the wheat plantlets to give 105 bacteria per mL. Germination, inoculation and growth of plantlets Seeds of Triticum aestivum (cv. CD104) were disinfected as described previously (Dobereiner, Baldani e Baldani, 1995; (Camilios-Neto et al. 2014). In vitro plant cultivation was done under hydroponic and axenic conditions. Surface sterilized seeds were pre-germinated in agar/water petri dishes in the dark for 24 h at 30°C. The plantlets were then transferred to glass test tubes (two plantlets in each tube) containing 25 mL of Hoagland’s medium and 10 cm3 of polypropylene spheres were added to each tube to serve as support for the plantlets (Fig. S1). On the second day after germination, plantlets were inoculated with H. seropedicae to a final density of 105 H. seropedicae cells per mL of Hoagland’s medium.	The rRNA was depleted using the Microbe Express kit (Ambion®), the libraries for sequencing were prepared using the Whole Transcriptome Analysis kit (Life Technologies).	Attached bacteria were recovered by vigorous vortexing of roots from 80 plantlets, 3 days after inoculation in Hoagland’s medium followed by centrifugation. Planktonic cells were collected by centrifugation from the hydroponic medium at the same time. The pellet was resuspended in RNA latter (Ambion®) and stored at - 20oC. The total RNA of H. seropedicae cells (~ equivalent to 106 CFU/g of fresh wheat root) was extracted using Trizol (Invitrogen®) and treated with DNAseI (Ambion®) following the manufacturer instructions. The
Protocol Parameters					
Protocol Hardware		AB SOLiD 3 Plus System			
Protocol Software	CLC Workbench and DESeq				
Protocol Contact					

Term Source Name	EFO	ArrayExpress
Term Source File	http://www.ebi.ac.uk/efo/	http://www.ebi.ac.uk/arrayexpress/
Term Source Version	2.36

SDRF File	E-MTAB-3646.sdrf.txt
Comment [Submitted Name]	RNA-seq Transcriptional Profiling of Herbaspirillum seropedicae Colonizing Bread Wheat (Triticum aestivum) Roots.
Comment [SecondaryAccession]	ERP012641
Comment [SequenceDataURI]	http://www.ebi.ac.uk/ena/data/view/ERR1049832-ERR1049841
Comment [AEExperimentType]	RNA-seq of coding RNA
Comment[ArrayExpressAccession]	E-MTAB-3646
Comment [AEMINSEQEScore]	3