title: a16
organism: Pectobacterium atrosepticum
description: Protocols: After 3 hours incubation at 20C, 20ml cultures were centrifuged 4000rpm 10 minutes and the pellets flash frozen in liquid nitrogen. Radish (Raphanus sativus L. var. oleiformis Pers. cv. Bento) seeds were surface sterilised in 2% calcium hypochlorite solution (10ml) before germination on distilled water agar (0.5% w/v) in the dark for 5-7 days, at ~20C. Potato (Solanum tuberosum var. Estima) microplants (Gentech Propagation, UK) were cut at the internode, transplanted onto Murashige and Skoog (MS; Merck) + 20% (w/v) sucrose (MS20) agar and incubated for 14 days to establish new microplants. Seedlings, or microplants, were transplanted into 175ml pots (Greiner, UK) containing autoclaved perlite and sterile 0.5 x MS liquid medium. Seedlings were grown 16 h light/ 8h dark photoperiod for a further seven (potato) or 21 days (radish) at ~20C before Pectobacterium atrosepticum SCRI1039 inoculation. Treatments ended after 14 days. Each biological sample pooled the roots from three radish or two potato plants. Three biological replicate cultures of Pectobacteria atrosepticum were grown in Luria Broth overnight at 27 degrees Celsius, 150rpm. The bacteria were washed in 0.5x MS media and diluted to a final concentration of 10^8 cfu/ml in 0.5x MS media. Total RNA was extracted using Macharey-Nagel Nucleospin RNA Plant kit, following manufacturers guidelines with RAP buffer. Samples were treated with Turbo DNase-free kit (Invitrogen, AM1906) to remove gDNA carryover. RNA quality was checked with Agilent Bioanalyzer 2100 following supplier´s instructions RNA was extracted using the Macherey Nagel Plant RNA kit with the following modifications, the frozen bacterial pellet was resuspended in 100ul TE + 1mg/ml lysozyme (Sigma, UK). After incubating at 37 degrees Celsius for 10 minutes, 600ul of RA1 buffer was added, the sample vortexed and 600ul 70% ethanol added before continuing the manufacturer's protocol. Library construction was completed by Novogene.150bp paired end Illumina library
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2024-01-17T00:28:37Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2024-01-17T00:28:37Z
attributes.tag: External Id
attributes.value: SAMEA114498705
attributes.tag: INSDC center name
attributes.value: Information and Computational Sciences The James Hutton Institute
attributes.tag: INSDC first public
attributes.value: 2024-01-17T00:28:37Z
attributes.tag: INSDC last update
attributes.value: 2024-01-17T00:28:37Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-13434:a16
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: collection date
attributes.value: not collected
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: infect
attributes.value: not applicable
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: sample name
attributes.value: E-MTAB-13434:a16
attributes.tag: scientific_name
attributes.value: Pectobacterium atrosepticum
attributes.tag: strain
attributes.value: SCRI1039