Rhodococcus RNA-Seq: under E2 conditions
Briefly, the rRNA was depleted from 1 microgram of total RNA using the Ribo-ZeroMagnetic Gold Kit. TruSeq RNASample Prep Kit v2 was used for libraryconstruction. RNAwas fragmented into small pieces using Elute Prime Fragment Mix.First-strand cDNA was synthesized with First Strand Master Mix and Super Script II reverse transcription. After product purification with Agencourt RNAClean XPBeads , the second-strand cDNA library wassynthesized using Second Strand Master Mix and dATP, dGTP, dCTP, dUTP mix. Purified fragmented cDNA was end repaired and purifiedwith AMPureXP Beads .Addition of the poly (A) tailwas done with A-tailing Mix prior to ligating sequencing adapters. The second-strand cDNA was degraded using theUracil-N-Glycosylase enzyme and the product was purified byAMPureXP Beads. Several rounds of PCR amplification with PCR Primer Cocktailwere performed to enrich the cDNA fragments and the PCR products were purifiedwithAMPureXPBeads.