title: RNAseq of Neisseria gonorrhoeae FA19 penA41 +- acnB-G348D: mid-log phase growth in GCB
design: Liquid cultures were grown until the cells reached log-phase (OD600~0.4-0.5). Three ml were removed, the cells were pelleted, resuspended in RNA Later, and stored overnight at 4oC. The next morning, total RNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen, Carlsbad, CA), contaminating DNA was removed by DNase digestion using the Invitrogen TURBO DNA-free Kit, and the RNA was reisolated using the RNeasy Mini Kit. Ribosomal RNA was removed by 2 successive rounds of treatment with the Life Technologies MICROBExpress mRNA prep kit (ThermoFisher, Grand Island, NY), and the mRNA was recovered with the Qiagen RNeasy MinElute Cleanup Kit. All mRNA samples were analyzed using the Agilent High Sensitivity ScreenTape system to determine quality (Agilent, Santa Clara, CA) and by Nanodrop to assess quantity. Libraries were prepared using the Illumina TruSeq High Sample (HS) Protocol  (Illumina, San Diego, CA). First and second strand cDNA were synthesized using AMPure XP magnetic beads, first and second strand master mixes, and SuperScript II Reverse Transcriptase. End repair, 3'end adenylation, adapter ligation, and DNA fragment enrichment were all performed according to the TruSeq protocol. The quality of the DNA libraries was analyzed by the Agilent High Sensitivity ScreenTape system and the concentrations were determined using the Qubit dsDNA HS Assay kit (ThermoFisher, Grand Island, NY). Equimolar DNA amounts of each library were pooled and prepared for sequencing on the Illumina NextSeq 500 System according to the manufacturers system guide.