characteristics: ERC000011
characteristics: ENA-CHECKLIST
characteristics: 2022-12-15T00:25:58Z
characteristics: ENA-FIRST-PUBLIC
characteristics: 2022-12-15T00:25:58Z
characteristics: ENA-LAST-UPDATE
characteristics: SAMEA110177421
characteristics: External Id
characteristics: Department of Molecular Microbiology John Innes Centre
characteristics: INSDC center name
characteristics: 2022-12-15T00:25:58Z
characteristics: INSDC first public
characteristics: 2022-12-15T00:25:58Z
characteristics: INSDC last update
characteristics: public
characteristics: INSDC status
characteristics: ERS12276227
characteristics: SRA accession
characteristics: E-MTAB-11868:SB0_k2
characteristics: Submitter Id
characteristics: ArrayExpress
characteristics: broker name
characteristics: Protocols: A single colony from each conjugation event was picked and grown overnight in 10 mL KB medium with Gentamicin and Kanamycin at 28 degree Celsius, 230 rpm. A single colony from each conjugation event was picked and grown overnight in 10 mL KB medium with Gentamicin and Kanamycin at 28 degree Celsius, 230 rpm. The OD600 of these cultures was measured and 60 mL KB cultures were set up at OD600 = 0.01. Cultures were grown at 28 degree celsius, 230 rpm until OD600 = 1.4. The .bam files produced by aligning the reads to the genome were used as input to the featureCounts tool of the Bioconductor package Rsubread (version 2.0.0) along with a a .saf file containing coordinates of genes in the genome. This produced a table of counts of reads mapping to every gene in the genome. 2 mL of culture was harvested for RNA extraction. Pellets were collected at 8000 xg, 10 minutes at 4 degree Celsius and flash frozen in liquid nitrogen before storage at -80 degree Celsius. Pellets were resuspended in 150 µL 10 mM Tris-HCl pH 8 and mixed with 700 µL of ice cold RLT+BME (RLT buffer (Qiagen) supplemented with 1% β-mercaptoethanol) and cells were lysed using a Fastprep (MP Bio) using Lysis matrix B beads (MP Bio). Lysis matrix was removed by centrifugation (13,000 xg, 3 minutes) and the supernatant was added to 450 µL of ethanol. The supernatant was applied to a RNeasy column and RNA extraction was performed as per the manufacturer’s instruction including the on-column DNA digest. After extraction a Turbo DNase (Promega) digest was performed as per the manufacturer’s instruction and total RNA yield was quantified using a QBIT. Library construction was carried out by an external contractor (Novogene) using the Illumina RNA-Seq library preparation kit.
characteristics: description
characteristics: wild type genotype
characteristics: genotype
characteristics: KB medium
characteristics: growth condition
characteristics: Pseudomonas fluorescens
characteristics: organism
characteristics: Pseudomonas fluorescens
characteristics: organism
characteristics: whole organism
characteristics: organism part
characteristics: Pseudomonas fluorescens
characteristics: scientific_name
characteristics: SBW25
characteristics: strain
characteristics: SB0_k2
characteristics: title