GSM1482975: T120_1; Mycolicibacterium smegmatis; Mycobacterium phage Kampy; RNA-Seq
15 ml of infected M. smegmatis culture was pelleted by centrifugation for 10 minutes at 400 G at 4°C. Pellets were flash frozen in liquid nitrogen and stored at -80°C until further processing. Frozen cell pellets were resuspended in 1 ml TRIzol (Life Technologies). Cells were then disrupted by beadbeating twice for 45 seconds, with one minute on ice between cycles. Total RNA was extracted from the TRIzol solution with bromochloropropane, washed with isopropanol, precipitated with 70% ethanol, and resuspended in nuclease-free water. Total RNA was incubated with recombinant DNase I (Ambion) and RNasin (Promega) for 30 minutes at 37°C. DNase I was inactivated by addition of EDTA (5 mM) and 10 minute incubation at 70°C. Ribosomal RNA was depleted from prepared samples by subtractive hybridization using the RiboZero kit for gram-positive bacteria (Epicentre) and depleted RNA was checked for integrity and rRNA depletion with the Bioanalyzer 2100 (Aligent). Libraries for sequencing were prepared from rRNA-depleted RNA using the Ion Total RNA-Seq Kit v2 (Ion Torrent) and sequenced on an Ion Torrent PGM according to the manufacturer's instructions.
GEO Accession
GSM1482975