Burkholderia cenocepacia
Protocols: B. cenocepacia growth in NGMII medium was collected with M9 buffer and then preserved in Trizol reagent Total RNA extraction was performed with the reagent TRIzol™ following the manufacturer's instructions. The bacterial RNA enrichment was achieved using the Cappable-seq for prokaryotic transcription start site (TSS) determination (New England Biolabs®Inc., NEB). This method consists of capping the 5' triphosphorylated RNA with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) (NEB) using the vaccinia capping enzyme (VCE) (NEB) for reversible binding of biotinylated RNA species to streptavidin. The Cappable-seq enriched RNAs were first poly(A)-tailed using poly(A) polymerase. Then, the 5'PPP structures were converted into 5' monophosphate ends using CapClip Acid Pyrophosphatase (Cellscript). Afterwards, an RNA adapter was ligated to the 5'-monophosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified to about 10-20 ng/μl using a high-fidelity DNA polymerase.The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). The cDNA pool was size fractionated in the size range of 200 - 550 bp using a preparative agarose gel. The 3' and 5' adapters and the primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina.