6B
Staphylococcus aureus
Protocols: Cultures of S. aureus ATCC 51811 containing the appropriate pRAB11 overexpression or control plasmid were grown with shaking at 37degreesC for 50 min, then expression of sigH/comK1/comK2 was induced by addition of 4uM anhydrotetracycline after 50 min and cultures were grown for 3 hours in total. Cells were harvested by incubating cultures in an equal volume of ice-cold methanol for 3 min before centrifugation at 4000g for 10 min at 4degreesC. Pellets were frozen on liquid nitrogen and stored at -80degreesC. Two independent biological replicates were collected for each of the six strains. Cells were lysed using Lysing Matrix B and a FastPrep instrument (both MP Biomedicals). RNA was isolated using the RNeasy Mini Kit (Qiagen). RNA was treated with TURBO DNase (Ambion) as described, purified using the RNA Cleanup protocol from the RNeasy Mini Kit (Qiagen), and stored at -80ï¾°C. The integrity of the RNA was analyzed using an Agilent 2100 Bioanalyzer (Agilent technologies). The RiboZero rRNA Removal Kit (Epicentre) was used twice to remove 23S, 16S, and 5S rRNA. The libraries were constructed using a strand-specific library construction protocol, and fragments of size range 100-500 bp were used to create 12 indexed libraries which were subsequently pooled. This step was performed by a commercial company.
strain
ATCC 15811
ENA-FIRST-PUBLIC
2014-09-04T17:01:10Z
organism
Staphylococcus aureus subsp. aureus
ENA-LAST-UPDATE
2014-03-10T11:22:09Z
scientific_name
Staphylococcus aureus subsp. aureus
genotype
pRAB11-comK2-sigH overexpression plasmid
ENA-FIRST-PUBLIC
2014-09-04
ENA-LAST-UPDATE
2014-03-10