Illumina HiSeq 1000 paired end sequencing; BCAM0258 overexpression mutant
BCAM0258 overexpression mutant
an inoculum suspension containing approximately 5 x 107 CFU/ml was added to the wells of a round-bottomed 96-well microtiter plate (TPP). Ten wells were included per strain and the experiment was repeated twice (n = 3 x 10). Biofilms were grown in LBB supplemented with Tp and 0.2 % rhamnose to induce toxin expression. Following 4h of adhesion, the supernatant was removed and the plates were rinsed with physiological saline (PS). Subsequently, 100 ul of fresh LBB supplemented with Tp and 0.2 % rhamnose was added and the plates were further incubated at 37degreesC. after 24 h of growth, cells were harvested by vortexing (5 min) and sonication (5 min) and transferred to sterile tubes. RNA was extracted immediately following harvesting of the cells using the Ambion RiboPure Bacteria Kit (Life Technologies, Renfrewshire, UK), according to the manufacturerﾒs instructions. This procedure included a DNase I treatment for 1 h at 37degreesC. RNA quantifications were performed using picogreen fluorescence measurement on a FLUOstar galaxy plate reader (BMG Labtech, Ortenberg, Germany). Two microgram total RNA of each sample was depleted for ribosomal RNA using the Ribo-zero Magnetic Gram-Negative Bacteria kit (Epicentre, Madison, WI, U.S.A.). Approximately 30 ng of rRNA depleted RNA was used to create barcoded strand specific cDNA sequencing libraries with the Truseq stranded Total RNA library preparation kit (Illumina, San Diego, USA).
GHENT UNIVERSITY
E-MTAB-2079:assay mut3
Ghent University
E-MTAB-2079:assay mut3
Experimental Factor: genotype
BCAM0258 overexpression mutant