SBP_k4
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.
ENA-FIRST-PUBLIC
2022-12-15
ENA-LAST-UPDATE
2022-12-15
External Id
SAMEA110177428
INSDC center alias
Department of Molecular Microbiology John Innes Centre
INSDC center name
Department of Molecular Microbiology John Innes Centre
INSDC first public
2022-12-15T00:25:58Z
INSDC last update
2022-12-15T00:25:58Z
INSDC status
public
Submitter Id
E-MTAB-11868:SBP_k4
broker name
ArrayExpress
genotype
pQBR103
growth condition
KB medium
organism part
whole organism
sample name
E-MTAB-11868:SBP_k4
strain
SBW25