MAGE-TAB Version	1.1
Investigation Title	Ribosome profiling of Mycobacterium tuberculosis under nutrient starvation

Experimental Design	stimulus or stress design
Experimental Design Term Source REF	EFO
Experimental Design Term Accession Number	EFO:0001762

Experimental Factor Name	environmental stress
Experimental Factor Type	environmental stress
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	2020-02-24
Public Release Date	2021-02-02

PubMed ID
Publication DOI	10.1016/j.celrep.2021.108695
Publication Author List	Elizabeth B. Sawyer, Jody E. Phelan, Taane G. Clark and Teresa Cortes
Publication Title	A snapshot of translation in Mycobacterium tuberculosis during exponential growth and nutrient starvation revealed by ribosome profiling
Publication Status
Publication Status Term Source REF
Publication Status Term Accession Number

Experiment Description	We have performed parallel ribosome profiling and RNA sequencing to determine which mRNAs are being translated during exponential growth and following 24 hours of nutrient starvation in the human pathogen Mycobacterium tuberculosis.

Protocol Name	P-MTAB-94238	P-MTAB-94237	P-MTAB-94239	P-MTAB-94240
Protocol Type	nucleic acid extraction protocol	sample collection protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol
Protocol Term Source REF	EFO	EFO	EFO	EFO
Protocol Term Accession Number	EFO_0002944	EFO_0005518	EFO_0004184	EFO_0004170
Protocol Description	Sample preparation for ribosome profiling was carried out according to a modified form of the method of Latif et al.(Latif et al., 2015). All steps were performed on ice or using chilled buffers. M. tuberculosis cell pellets were resuspended in lysis buffer (20 mM HEPES, pH 7.4; 200 mM KCl, 1% TritonX-100, 12 mM MgCl2, 1 mM CaCl2, 1 mg/ml heparin, 100 μg/ml chloramphenicol and 5 U/ml DNase I)  and transferred to ribolysing matrix tubes (MPBio). Cell lysis was performed by ribolysing the sample three times for 30 s at power 6.5 (FastPrep-24, MPBio). Ribolysing matrix and cell debris were removed by centrifugation at 12,000 rpm (JA-15 1.5 rotor, Beckman) for 10 minutes.The ribosome profiling samples were then double filtered through a 0.2 m membrane (Corning) to ensure sterility before incubation for 45 minutes with 24 l microccocal nuclease (NEB) and 15 l DNaseI (Thermo Fisher) to digest mRNAs that are not bound by ribosomes . Nuclease digestion was stopped by addition of 10 l Superase In (Thermo Fisher) and 5 l 0.5 M EGTA and cell lysates loaded onto a 34% sucrose cushion. Ribosomes were harvested by centrifugation at 40,000 rpm for 15 h in an SW40Ti rotor (Beckman). To isolate the ribosome-bound mRNAs or ribosome footprints (RPF), the ribosomal pellet was then resuspended in Qiazol (Qiagen) and small RNA isolated using the miRNeasy mini kit (Qiagen). Isolated RNA was precipitated and resuspended in 10 mM Tris, pH 8.0. Finally, RPF samples were depleted of ribosomal RNA using the RiboZero rRNA removal kit for bacteria (Illumina) and purified using RNeasy MinElute columns (Qiagen). The quantity and quality of the RNA was assessed at each step using the bioanalyser and qubit.For RNA isolation, M. tuberculosis cell pellets were resuspended in RNApro solution (MPBio) and ribolysed three times for 30 s at power 6.5 (FastPrep-24, MPBio). Ribolysing matrix and cell debris were removed by centrifugation at 12,000 rpm (JA-15 1.5 rotor, Beckman) 4°C for 10 minutes. The total RNA sample was then removed from the ribolysing tube and chloroform added. The sample was vortexed for 10 s and incubated at room temperature for 5 minutes before centrifugation at 12,000 rpm (JA-15 1.5 rotor, Beckman) 4°C for 5 minutes. The aqueous phase was then transferred to a new tube and 1.5 volumes of 100% ethanol added. Samples were incubated at -20°C overnight and spun to pellet the RNA. The pellet was washed with 75% ethanol, air dried and resuspended in nuclease-free water. A further acid-phenol chloroform extraction was performed to improve purity of the RNA before resupending the final pellet in nuclease-free water. Genomic DNA contamination was assessed by PCR for 16S genomic DNA and, if present, samples were treated with DNase I followed by a further acid-phenol chloroform extraction.Next, samples were depleted of ribosomal RNA using the RiboZero rRNA removal kit for bacteria (Illumina) and purified using RNeasy MinElute columns (Qiagen). Finally, total RNA samples were then subjected to fragmentation using RNA fragmentation reagents (Thermo Fisher) at 80°C for 20 minutes. The quantity and quality of the samples were assessed at each step using the bioanalyser and qubit.	M. tuberculosis H37Rv was grown in Middlebrook 7H9 medium (BD Diagnostics) supplemented with 10% Middlebrook albumin-dextrose-catalase (ADC) (BD Diagnostics), 0.2% glycerol and 0.05 % Tween 80 at 37°C until OD600 was 0.4–0.6. For starvation experiments, exponentially growing bacteria were pelleted, washed twice in PBS and resuspended in PBS supplemented with 0.025% Tyloxapol, and maintained in culture for a further 24 h. Triplicate cultures of exponentially growing and nutrient starved cells were prepared for parallel RNA and Ribosome profiling experiments. For ribosome profiling experiments, chloramphenicol was added to a final concentration of 100 μg/ml two minutes prior to harvesting cells.	RPF and total RNA samples were converted to cDNA libraries as previously described (Latif et al 2015). Briefly, samples were treated with T4 PNK (NEB) at 37ºC for 30 mins and purified using RNeasy MinElute columns (Qiagen). The NEBnext small RNA library prep kit (NEB) was used according to manufacturer’s guidelines to construct the multiplexed cDNA libraries	Multiplexed sequencing was performed by Cambridge Genomic Services using a NextSeq 500 (Illumina) with single end reads of 75 bp.
Protocol Parameters
Protocol Hardware				NextSeq 500
Protocol Software
Protocol Contact

Term Source Name	EFO	ArrayExpress
Term Source File	http://www.ebi.ac.uk/efo/efo.owl	http://www.ebi.ac.uk/arrayexpress/
Term Source Version	2.38

SDRF File	E-MTAB-8835.sdrf.txt
Comment [TemplateType]	High-throughput sequencing
Comment [Submitted Name]	Ribosome profiling of Mycobacterium tuberculosis under nutrient starvation
Comment [SecondaryAccession]	ERP120307
Comment [SequenceDataURI]	http://www.ebi.ac.uk/ena/data/view/ERR3971466-ERR3971477
Comment [AEExperimentType]	Ribo-seq
Comment[ArrayExpressAccession]	E-MTAB-8835