title: Epiphytic
organism: Herbaspirillum rubrisubalbicans M1
description: Protocols: Herbaspirillum rubrisubalbicans M1 was grown at 30ºC and 120 rpm in NFbHPN medium (Klassen et al., 1997). Seeds of susceptible cultivar Sorghum bicolor cv. A07 were surface-sterilized, germinated on water-agar (0.8%) plates and transferred to a hydroponic system composed of 30 mL of plant medium (Egener et al., 1999 without carbon and nitrogen sources) and 10 g of polypropylene spheres in 100 mL glass tubes (Balsanelli et al., 2013). Each seedling was then inoculated with 108 CFU of H. rubrisubalbicans. In the control condition without a plant, the hydroponic medium was supplemented with 2 mM of malate. Bacterial samples were taken 3 days after inoculation in duplicate. Free-living bacteria of the samples without plants (named “control”) were isolated from 250 mL of hydroponic medium by centrifugation (10,000 g, 8 min, 4°C) and re-suspended in cold lysis buffer (Tris 50 mM pH 8.0, EDTA 10 mM, SDS 0.6%). Epiphytic bacteria were removed from roots by vortexing 10 g of roots in RNAlater (Sigma) during 5 min, isolated by centrifugation (10,000 g, 8 min, 4°C) and re-suspended in cold lysis buffer. For both samples (each in duplicate), total RNA was purified using TRI® Reagent (Sigma) followed by chloroform extraction as suggested by the manufactor. Samples were treated with turbo DNase (Ambion) and the RNA quantified with a NanoDrop spectrophotometer (THERMO Scientific) and Bioanalyzer 2100 (Agilent). The ribosomal RNA was partially depleted from 2 μg of total RNA using the Ribo Zero Bacteria kit (Epicentre). The indexed cDNA libraries were constructed from 0.2 μg ribosomal-depleted RNA samples using the TruSeq Stranded RNA Sample Preparation kit v3 (Illumina).
attributes.tag: ENA first public
attributes.value: 2015-08-13
attributes.tag: ENA last update
attributes.value: 2015-04-30
attributes.tag: External Id
attributes.value: SAMEA3367587
attributes.tag: INSDC center alias
attributes.value: UFPR
attributes.tag: INSDC center name
attributes.value: UFPR
attributes.tag: INSDC first public
attributes.value: 2015-08-13T17:01:07Z
attributes.tag: INSDC last update
attributes.value: 2015-04-30T09:56:40Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-3538:Epiphytic
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: growth condition
attributes.value: Sorghum root epiphytic cells
attributes.tag: sample name
attributes.value: E-MTAB-3538:Epiphytic