title: Illumina MiSeq sequencing; RNA-seq of differential gene expression in response to teixobactin in Enterococcus faecalis
design: RNA-seq of differential gene expression in response to teixobactin in Enterococcus faecalis
library_construction_protocol: Two cultures of E. faecalis were grown to an OD600 of 0.2 and subsequently split into three to form a total of six cultures (two samples, with three technical replicates each), and grown to an OD600 of 0.5. One set of three cultures were treated with 0.5 ug/ml (0.25 x MIC), and all six cultures were grown for a further 1h. Cells were harvested by centrifugation and stored at -80 degrees C. Cells were harvested by centrifugation in 3:2 glycerol saline at 1Ok x g for 20mins at -10 degrees C. Cells were resuspended in 1:1 glycerol saline, snap frozen and stored at -80 degrees C for RNA extraction the following day. Two 100ml Schott bottles containing 62.5ml of brain heart infusion (BHI) broth were inoculated 1/100 with E. faecalis JH2-2 overnight culture and grown to an OD600 of  0.2 (37 degrees C with gentle agitation). Each culture was split into triplicate and grown to an OD600 of 0.5. One set of triplicates were treated with 0.5ug/ml of teixobactin, while the other set of triplicates were an untreated control. Cells were grown for a further 1 h and harvested as stated above. RNA was extracted by TRIzol-chloroform as per manufacturers instructions. Residual DNA was removed from samples using the Ambion TURBO DNA-free kit. RNA was further purified using the RNeasy Mini Kit (Qiagen) as per the manufacturers instructions.  The Illumina TruSeq stranded total RNA library prep kit and ribosomal RNA was removed using Ribo-Zero.
attributes.tag: Experimental Factor: compound
attributes.value: teixobactin
attributes.tag: Experimental Factor: dose
attributes.value: 0.5