Instrument: Ion Torrent Proton
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Immediately after obtaining the cell pellet, 100 µl of killing buffer was added. Mechanical cell disruption was carried out with a Teflon vessel and a disruption ball filled with liquid N2 and pre-cooled in liquid N2 . The resulting cell powder was resuspended in 4 ml of lysis solution by repeated pipetting and transfer into 1 ml aliquots. Subsequently, one volume of acid phenol solution was added to cell lysate, and mixed on a tube shaker. Centrifugation and supernatant transfer into a new tube were followed by the addition of one volume acid phenol solution. This procedure was repeated twice; first by the addition of one volume chloroform/IAA and then by the addition of Na-Acetate and isopropanol for overnight precipitation. RNA extraction was carried out by centrifugation at 4°C, where the pellet was washed twice with 80% ethanol, dry at room temperature, and dissolved in nuclease-free water. DNase Digestion and RNA clean-up (Qiagen, Hilden, Germany) were performed following manufacturer's protocol. RNA concentration was determined by Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and RNA quality assessment was carried out by Agilent 2100 Bioanalyzer. cDNA libraries were constructed using the Ion Total RNA-Seq Kit v2 (Life Technologies, Carlsbad, CA) and the manufacturer's recommended protocol.