GSM1678900: Ex vivo2; Glaesserella parasuis str. Nagasaki; RNA-Seq
Sequencing library was generated using an Ion Torrent RNA-Seq v2 kit (Life Technologies) and RNA was sequenced using an Ion Torrent PGM instrument (Life Technologies) with an Ion 316 chip (Life technologies) at the Centre for Research in Agricultural Genomics (CRAG, Campus de Bellaterra-UAB, Spain). As control, RNA from the Nagasaki strain grown overnight on chocolate agar plates was purified and sequenced in the same manner. Bacteria were recovered by centrifugation and pellets were used for RNA extraction. An additional low speed centrifugation step was added to ex vivo and in vivo samples in order to eliminate mammalian cells. All bacterial samples were processed for RNA extraction as follows. Pellets were resuspended in 2% SDS 16 mM EDTA 10 mM TRIS (pH XX) and incubated for 5 min at 100 ºC. Afterwards, samples were processed by two hot acid phenol-chloroform extractions, followed by two chloroform/ isoamyl alcohol extractions. RNA was then precipitated with 0.6 volumes of isopropanol 0.1 volumes of 5M ammonium acetate and 1μL of glycogen. After centrifugation the pellet was washed with 70% ethanol, dried and resuspended in warmed RNase-free water. To ensure that contaminating bacterial DNA was eliminated from the samples, treatment with RNase-free DNase (Qiagen) was performed. In addition, ribosomal RNA was eliminated with the Ribo-Zero rRNA removal kit (Epicentre Biotechnologies, Madison, WI, USA) following manufacturer's instructions. PCR reactions using primers specific for H. parasuis 16S rRNA gene were carried out to ensure that no bacterial DNA was left in the sample.
GEO Accession
GSM1678900