title: GSM5104842: WT_TB47_3_L8; Mycolicibacterium smegmatis; RNA-Seq
library_construction_protocol: Total RNA was extracted using TRIZOL® reagent (Invitrogen) according to the manufacturer's instructions. Cell lysis was achieved by three cycles of bead-beating in a Mini-Beadbeater (Biospec) at 5,000 rpm for 30 sec. DNA was removed from the RNA preparation by treatment with 2 U RNase-free DNase using the TURBO DNA-free kit (Ambion), according to the manufacturer's instructions. RNA concentrations were determined using a NanoDrop® ND-1000 spectrophotometer and the quality of RNA checked on a 1.2% agarose gel. Library preparation and sequencing of the M. smegmatis transcriptome was carried out by the Otago Genomics and Bioinformatics Facility (OGBF) in conjunction with New Zealand Genomics Limited (NZGL). Prepared RNA was checked for quality on a Bioanalyzer (Agilent), followed by ribosomal RNA depletion with the Bacterial Ribo-zero RNA Removal Kit (Illumina). Quantification of nucleic acids was performed using a Qubit fluorometer® (Thermo Fisher Scientific). Libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit (Illumina), before being sequenced across two lanes on an Illumina HiSeq with paired-end read lengths of 125 base pairs and using the v4 chemistry.