GSM2045104: Nitrogen excess condition, exponential phase 1; Rhodococcus jostii; RNA-Seq
Total RNA was extracted using TRIzol® Reagent (Invitrogen) according to the manufacturer’s instructions. The RNA was treated with TURBOTM DNase (Invitrogen) and extracted with phenol-chloroform. The quality and concentration of RNA were assessed using a 2100 Bioanalyzer and the RNA 6000 Nano kit (Agilent). Three µg of total RNA was heated at 65 °C for 5 min, quickly chilled on ice and then mixed with Streptavidin magnetic beads (New England Biolabs) with an equimolar mixture of biotin-labelled probes specific for RHA1 rRNA (23S: AGGAATTTCGCTACCTTAGGATGGTTATAGTTACC and CTTTTC- ACCTTTCCCTCACGGTACTAGTCCGCTAT; 16S: ATATGTCAAACCCAGGTAAGGT- TCTTC) to deplete rRNA. The supernatant was collected and subjected to further rRNA-depletion using the Ribominus™ Transcriptome Isolation Kit (Invitrogen). One hundred ng of the rRNA-depleted RNA was used for library construction using NEBNext® Ultra Directional RNA Library Prep Kit for Illumina® and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs) according to the manufacturer’s instructions. The samples were analyzed using a Genome Analyzer IIx (Illumina®).
GEO Accession
GSM2045104