Bacterial RNA Extraction and Sequencing

Total RNA was extracted from the stored RNAprotect solution using an RNAeasy Qiagen kit (Qiagen, Valencia, CA) as indicated by the manufacturer’s instructions, followed by an additional treatment with DNase I (Qiagen, Valencia, CA) to remove traces of DNA. rRNA depletion and mRNA enrichment were performed using an Illumina Ribo-Zero rRNA removal kit (catalog no. MRZ116C) according to the instructions of the manufacturer (Illumina, San Diego, CA). RNA concentrations were measured using an RNA Qubit assay (Invitrogen, Burlington, Ontario, Canada), and RNA integrity was evaluated using a Bioanalyzer RNA Pico assay (Agilent Technologies, Santa Clara, CA) (18 (link)). cDNA libraries were constructed using a ScriptSeq Complete kit for bacteria (Epicentre, Madison, WI). cDNA was then purified using an Agencourt AMPure XP system (Beckman Coulter, Beverly, MA), and the second cDNA was generated by adding the Illumina adapters as the forward primer and a ScriptSeq index primer as the reverse primer. The resulting libraries were purified using an AMPure XP system (Beckman Coulter, Beverly, MA), quantified with the DNA Qubit assay, and evaluated using the Bioanalyzer sensitivity DNA assay (Agilent Technologies, Santa Clara, CA). One hundred single reads were generated using the Illumina HiSeq 2000 platform at the National Center for Genome Research in Santa Fe, NM.