title: AB SOLiD 4 System sequencing; Herbaspirillum seropedicae transcript profile on maize rhizosphere
design: Herbaspirillum seropedicae transcript profile on maize rhizosphere
library_construction_protocol: H. seropedicae SmR1 was grown at 30oC and 120 rpm in NFbHPN medium (Klassen et al., 1997) in the presence of streptomycin 80 ug.mL-1. Assays of maize colonization by H. seropedicae were performed according to Balsanelli et al. (2013). Briefly, seeds of Zea mays cv. 30F53 were surface-sterilized, germinated and each seedling was inoculated with 10^8 CFU of H. seropedicae. The inoculated seedlings were transferred to glass tubes containing 30 mL of plant medium (Egener et al., 1999) without carbon and nitrogen sources and 10 g of sterile polypropylene beads. Bacterial samples were taken in duplicate 1 and 3 days after inoculation. Free-living bacteria (planktonic, samples P) were isolated from 1 L of hydroponic medium by centrifugation (10,000 g, 8 min, 4oC) and re-suspended in cold lysis buffer (Tris.HCl 50 mM pH 8.0, EDTA 10 mM, SDS 0.6%). Epiphytic (samples E) bacterial cells were removed from roots by vortexing 30 g of roots in 20 mL of buffered saline during 1 min, harvested by centrifugation (10,000 g, 8 min, 4oC) and re-suspended in cold lysis buffer. For both samples, total RNA was purified using TRI Reagent (Sigma) followed by chloroform extraction. Samples were treated with DNaseI (Ambion) and the RNA quantified with NanoDrop (THERMO Scientific) and gel electrophoresis. The ribosomal RNA was partially depleted using the Microbe Express kit (Ambion) twice. The cDNA libraries were constructed from 0.5 ug ribosomal depleted RNA samples using the SOLiD Whole Transcriptome Analysis Kit (Life Technologies) and were barcoded using the SOLiD Transcriptome Multiplexing Kit (Life Technologies). SOLiD ePCR Kit and SOLiD EZBead Enrichment Kit (both Life Technologies) were used for processing the samples for sequencing.
attributes.tag: Experimental Factor: growth condition
attributes.value: Maize root surface
attributes.tag: Experimental Factor: time
attributes.value: 1 day
attributes.tag: LIBRARY_STRAND
attributes.value: not applicable