MAGE-TAB Version 1.1 Investigation Title Time course RNA-seq of Mycobacterium tuberculosis exposed to nitric oxide. Experimental Design stimulus or stress design Experimental Design Term Source REF EFO Experimental Design Term Accession Number EFO_0001762 Experimental Factor Name compound time Experimental Factor Type compound time Experimental Factor Term Source REF Experimental Factor Term Accession Number Person Last Name Cortes Person First Name Teresa Person Mid Initials Person Email teresa.cortes@lshtm.ac.uk Person Phone Person Fax Person Affiliation London School of Hygiene and Tropical Medicine Person Address Keppel Street WC1E 7HT London Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Quality Control Type Quality Control Term Source REF Quality Control Term Accession Number Replicate Type Replicate Term Source REF Replicate Term Accession Number Normalization Type Normalization Term Source REF Normalization Term Accession Number Date of Experiment 2017-02-22 Public Release Date 2017-08-29 PubMed ID 28811595 Publication DOI 10.1038/s41598-017-08306-1 Publication Author List Cortes T, Schubert OT, Banaei-Esfahani A, Collins BC, Aebersold R, Young DB. Publication Title Delayed effects of transcriptional responses in Mycobacterium tuberculosis exposed to nitric oxide suggest other mechanisms involved in survival. Publication Status Publication Status Term Source REF Publication Status Term Accession Number Experiment Description Mycobacterium tuberculosis is an intracellular human pathogen with the ability to resist and adapt to many adverse conditions it encounters upon infection. Among these, overcoming the production of nitric oxide by macrophages could be key for M. tuberculosis success. We have challenged M. tuberculosis with a sub-lethal concentration of nitric oxide and followed the transcriptomic response through RNA-seq for 48 hours. Protocol Name P-MTAB-54408 P-MTAB-54409 P-MTAB-54413 P-MTAB-54414 P-MTAB-54412 P-MTAB-54411 P-MTAB-54410 Protocol Type growth protocol treatment protocol high throughput sequence alignment protocol normalization data transformation protocol nucleic acid sequencing protocol nucleic acid library construction protocol nucleic acid extraction protocol Protocol Term Source REF EFO EFO EFO EFO EFO EFO EFO Protocol Term Accession Number EFO_0003789 EFO_0003969 EFO_0004917 EFO_0003816 EFO_0004170 EFO_0004184 EFO_0002944 Protocol Description 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 37C). 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. Raw reads were first filtered to discard low quality reads. FastQC (Babraham Bioinformatics) was used to inspect read base quality scores. Poor quality read bases were trimmed using the SolexaQA package; default parameters were used, trimming bases with confidences p > 0.05, and removing reads < 25 bases. Reference based assembly using the reference genome of M. tuberculosis H37Rv [EMBL:AL123456] was performed with BWA Normalised reads for each library were computed using DESeq2 RNA-seq libraries were sequenced as single-end reads on an Illumina HiSeq 2000 sequencer. For construction of directional RNA-seq libraries, Ribo-Zero and ScriptSeq v2 Kits (Epicentre) were used. Briefly, 5 g of total RNA were used for ribosomal RNA removal using the Ribo-Zero rRNA removal kit (Gram-Positive Bacteria) following manufacturers instructions. 50 ng of Ribo-Zero-treated RNA were used to construct cDNA libraries for RNA sequencing using the ScriptSeq v2 Kit. 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 manufacturers 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). Protocol Parameters Protocol Hardware Illumina HiSeq 2000 Protocol Software Protocol Contact Term Source Name EFO ArrayExpress Term Source File http://www.ebi.ac.uk/efo/ http://www.ebi.ac.uk/arrayexpress/ Term Source Version 2.38 SDRF File E-MTAB-5557.sdrf.txt Comment [Submitted Name] Time course RNA-seq of Mycobacterium tuberculosis exposed to nitric oxide. Comment [SecondaryAccession] ERP022074 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR1880934-ERR1880948 Comment [AEExperimentType] RNA-seq of coding RNA Comment[ArrayExpressAccession] E-MTAB-5557 Comment [AEMINSEQEScore] 3