title: Ti_5dpi_3
organism: Pseudomonas [fluorescens] SBW25
description: Protocols: Aerial parts of the plants were removed and 8 ml of PBS and 12 ml of RNALater were added. Root systems were vortexed for 10 min at 4°C to resuspend bacteria. Samples were filtered through four layers of muslin cloth and the filtrate was centrifuged at 170 x g at 4 °C to remove any residual vermiculite. The supernatant was transferred to a new bottle and centrifuged for 10 min 17000 x g and 4°C to pellet the bacterial cells. Cell pellets were then flash-frozen for further use. Germinated barley seedlings were placed into sterile 50 ml plastic tubes containing washed, medium grain vermiculite and rooting solution. Seedlings were grown for 7 days at 25 °C with 16 h light cycle, then inoculated with 1 ml of PBS washed Pseudomonas fluorescens SBW25 culture adjusted to OD (600nm) of 1.0. Cell pellets were resuspended in 400 µl of 10mM Tris-Cl 6 pH 8.0 and 700 µl of RLT buffer with β-mercaptoethanol and transferred to Matrix B tubes (MP Biomedicals, UK) and lysed with two 90 second pulses in a FastPrep machine (MP Biomedicals, UK) with 90 seconds rest on ice between them. Samples were centrifuged for 15 min at 16000 x g and 4°C, and RNA extracted from the supernatant with a RNeasy Mini kit (Qiagen, GE). On-column digestion was conducted with a RNAse-Free DNase Set (Qiagen, GE). RNA quality and concentration was checked by a nanodrop spectrophotometer and a Qubit 2.0 Fluorometer with dilution steps conducted where necessary. Samples were subjected to a second DNase treatment with a TURBO DNA-free Kit (Thermo Scientific, USA) and RNA integrity was confirmed on a 1 percent agarose gel. rRNA depletion and library construction were carried out by the external contractor Novogene (Hong Kong).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-05-15T00:14:50Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-05-15T00:14:50Z
attributes.tag: External Id
attributes.value: SAMEA113413034
attributes.tag: INSDC center name
attributes.value: Department of Molecular Microbiology John Innes Centre
attributes.tag: INSDC first public
attributes.value: 2023-05-15T00:14:50Z
attributes.tag: INSDC last update
attributes.value: 2023-05-15T00:14:50Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12918:Ti_5dpi_3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: rhizosphere of tipple variety of barley
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: sample name
attributes.value: E-MTAB-12918:Ti_5dpi_3
attributes.tag: scientific_name
attributes.value: Pseudomonas fluorescens SBW25