title: Illumina HiSeq 2500 sequencing; Comparison of mid-log transcriptomes of clinical isolates of Mycobacterium tuberculosis
design: Comparison of mid-log transcriptomes of clinical isolates of Mycobacterium tuberculosis
library_construction_protocol: Bacterial cultures of different M. tuberculosis strains were grown 100ml of modified 7H9 (see Medium details) to an OD600 of 0.5 (+/- 0.1) in 1l roller bottles at 37C. 20OD equivalents were harvested by centrifugation. Mycobacterium tuberculosis strains were grown in a modified 7H9 medium: we omitted oleic acid and Tween 80 from the medium, supplementing it instead with 0.05% w/v tyloxapol. Whole cell RNA was extracted from bacterial pellets by resuspending them in the RNApro solution (MP Biomedicals) and disrupting them by beadbeating for 40s with the MP biomedicals FastPrep cell homogeniser. All the reagents and tubes were purchased as the Fast RNA Blue kit from MP biomedicals. RNA was isolated with an acid phenol extraction. Whole cell RNA isolates were treated with Turbo DNAse from Ambion, the ribosomal rRNA was depleted using the RiboZero Gram-positive kit from Illumina. We used the TruSeq stranded total RNA library prepration kit from Illumina.