title: Illumina HiSeq 2000 sequencing; Time course RNA-seq of Mycobacterium tuberculosis exposed to nitric oxide.
design: Time course RNA-seq of Mycobacterium tuberculosis exposed to nitric oxide.
library_construction_protocol: Mycobacterium tuberculosis H37Rv (SysteMTb) was grown in Middlebrook 7H9 medium supplemented with 0.4% glycerol, 0.085% NaCl, 0.5% BSA and 0.05% Tyloxapol in roller bottle culture (2 rpm at 37°C).  For nitric oxide experiments, cultures were grown until mid-exponential phase and Diethylenetriamine/nitric oxide adduct (deta/NO, Sigma) was added to a final concentration of 1 mM.  For transcriptomic analyses, samples were harvested from triplicate cultures immediately before addition of nitric oxide and 20 minutes, 2 and 24 hours after nitric oxide addition. For each sample, 25 mL of culture were spun down and isolation of RNA was performed using the FastRNA Pro blue kit from QBiogene/MP Bio according to manufacturer’s instructions. Cultures were first rapidly cooled by addition of ice directly to the culture prior to centrifugation. All RNA samples were treated with Turbo DNase free (Ambion) until any DNA contamination removed. Concentration and quality control of RNA samples was measured by Nanodrop (ND-1000, Labtech) and Agilent RNA chip (2100 Bioanalyser). For construction of directional RNA-seq libraries, Ribo-Zero™ and ScriptSeq™ v2 Kits (Epicentre) were used. Briefly, 5 µg of total RNA were used for ribosomal RNA removal using the Ribo-Zero rRNA removal kit (Gram-Positive Bacteria) following manufacturer’s instructions. 50 ng of Ribo-Zero-treated RNA were used to construct cDNA libraries for RNA sequencing using the ScriptSeq™ v2 Kit.
attributes.tag: Experimental Factor: nitric oxide
attributes.value: compound
attributes.tag: Experimental Factor: 20 minutes
attributes.value: time