Illumina HiSeq 2500 sequencing; RNA-seq of Streptococcus pyogenes biofilms
RNA-seq of Streptococcus pyogenes biofilms
Overnight cultures of S. pyogenes 2001 were 1:30 diluted in a total volume of 4 ml THY in a 6 well plate (TPP Tissue culture plates, Sigma) Cultures were supplemented with 50 µg/ml SpeA or the same volume of PBS (pH 7.4) respectively and incubated for 2 hours at 37°C in the presence of 5% CO2.  Eight ml of RNAprotect (Qiagen) was subsequently added followed by incubation at room temperature for 10 min. The bacterial cells were pelleted by centrifugation at 5000×g for 6 minutes and RNA was isolated using TRIzol® (Thermo Fischer) as per manufacturer's instruction. DNA was removed using DNase treatment (Turbo DNA-free™ Kit, Ambion, Life Technologies). Quality and concentration of the isolated RNA was estimated using Bio-Analyzer (Agilent 2001, BioRad). rRNA was then removed using the Ribo-Zero rRNA removal kit (Epicentre, Illumina) as per the instructions in the kit. The cDNA library was generated using Script SeqTM  (Epicentre, Illumina) as per manufacturer's instructions.