title: NextSeq 550 sequencing; RNA-seq of Streptococcus anginosus in a mono- and multispecies biofilm
design: RNA-seq of Streptococcus anginosus in a mono- and multispecies biofilm
library_construction_protocol: S. anginosus LMG 14502 isolated from a human throat, P. aeruginosa DK2 is a CF isolate, S. aureus LMG 10147 is a human wound isolate S. anginosus LMG 14502 (throat isolate), P. aeruginosa DK2 (CF isolate), and S. aureus LMG 10147 (wound isolate) were cultured overnight at 37°C in Brain Heart Infusion broth (BHI) (Oxoid, Basingstoke, UK). Inoculum suspensions containing S. anginosus alone, or in combination with S. aureus and P. aeruginosa, were made in medium. 96-well microtiter plates were filled with the inoculum suspensions. After 4 hours of adhesion at 37°C, the supernatant was discarded and 100 µl fresh medium was added. Cells were allowed to grow for an additional 20 hours to form a mature biofilm. For each condition 3 independent samples were incubated. RNA was extracted immediately following harvesting of the biofilm cells via vigorously pipetting up and down, using the Ambion RiboPure Bacteria Kit (Ambion, Austin, TX) according to the manufacturer’s instructions. rRNA depletion (on 2 µg total RNA) was done using the Ribo-Zero Magnetic Kit Bacteria (Epicentre, Illumina).  Truseq stranded RNA library preparation kit (Illumina) was then used to create strand specific libraries. Quality control of the libraries (DNA 1000 chip, Agilent Technologies, Santa Clara, US) and a concentration assay (qPCR) was performed according recommendations provided by Illumina. Quality control of the libraries (DNA 1000 chip, Agilent Technologies, Santa Clara, US) and a concentration assay (qPCR) was performed according recommendations provided by Illumina.
attributes.tag: Experimental Factor: monospecies
attributes.value: growth condition