{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":[null],"organism":["Mycobacterium tuberculosis H37Rv"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16773"],"description":["(i) RNA-seq of M. tuberculosis H37Rv cultures grown in the presence of BVL3572S for 6 h compared to untreated controls.  (ii) Genome-wide M. tuberculosis H37Rv CRISPRi libraries were grown in the presence of ATc for 5 days prior to drug exposure. Cultures were treated with sub-MIC BVL3572S concentrations without amino acid or in the presence of L-His, L-Ala, or both. After 14 days outgrowth, genomic DNA were harvested from cultures for which BVL3572S concentrations were partially growth inhibitory and from untreated controls. Abundance of sgRNA was analyzed by deep sequencing. (iii) Genome-wide M. tuberculosis H37Rv transposon mutant librairies were treated with BVL3572S or untreated for 15 days followed by a 7-day antibiotic-free outgrowth. Genomic DNA were extracted, indexed, and sequenced."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample Collection - 10 mL of the treated and control cultures were harvested by centrifugation at 3,000 x g for 5 min.","Sequencing - cDNA libraries were sequenced using an Illumina NextSeq 500 system (Illumina Inc.) in high-output mode. All samples were multiplexed on a single flow-cell lane and sequenced in single-read sequencing mode with read lengths of 150 bp.","Sample Collection - Cultures (12 mL) were harvested by centrifugation at 3,000 rpm for 10 min.","Library Construction - Transposon-genome junctions were amplified by PCR as described by Payros et al. PLoS Pathogens 2021 (DOI: 10.1371/journal.ppat.1010020). An index specific to each sample was added during this PCR, allowing all samples to be pooled to create the Tn-seq library (TruSeqMC DNA Nano).","Sample Collection - Cultures were harvested by centrifugation at 4,000 x g for 5 min.","Growth Protocol - 25 mL cultures were grown at 37°C without agitation to an OD600 of 0.7.","Nucleic Acid Extraction - Genomic DNA was isolated from bacterial pellets using the CTAB‐lysozyme method. Pellets were washed in 1 ml PBS + 0.05% Tween-80 before being resuspended in 800 μl TE buffer (10 mM Tris pH 8.0, 1 mM EDTA) and 50 µl lysozyme (15 mg/ml) and incubated at 37 °C for 16 h. 70 μl of 10% SDS and 5 μl of proteinase K (20 mg/ml) were added, and samples were incubated at 65 °C for 30 min. Next, 200 μl of NaCl 2.5 M and 80 μl of 10% CTAB were added, and samples were incubated at 65 °C for 30 min. Finally, 750 μl of ice-cold chloroform was added and samples were mixed. After centrifugation at 16,000 × g for 5 min and extraction of the aqueous phase, samples were removed from the biosafety level 3 facility. Samples were incubated with 25 μg RNase A for 30 min at 37 °C, followed by extraction with phenol:chloroform:isoamyl alcohol (pH 8.0, 25:24:1, Thermo Fisher BP1752I-400), then chloroform. Genomic DNA was precipitated from the final aqueous layer with the addition of 0.015 volume of 4 M sodium acetate and 0.6 volume of isopropanol. Mixtures were incubated for at least 2 h at -20°C. DNA pellets were centrifuged at 16,000 × g for 30 min at 4 °C and washed twice with 750 μl 80% ethanol. Pellets were dried and resuspended with nuclease-free water. DNA was quantified using the Qubit dsDNA BR assay kit (ThermoFisher Scientific).","Sequencing - This library was sequenced by Illumina paired-end sequencing.","Nucleic Acid Extraction - The pellets were resuspended in 1 mL of RNApro (FastRNA Pro Blue Kit, MP biomedicals) and homogenized in impact-resistant 2 mL tubes containing 0.1 mm silica spheres (Lysing Matrix B, MP biomedicals) using a FastPrep FP120 cell disrupter (Thermo Fisher Scientific) at 6.0 Hz for 40 s. The lysates were centrifuged at 12,000 x g and RNA was purified according to the manufacturer's instructions. Ribosomal RNA (rRNA) was depleted using the QIAseq FastSelect 5S/16S/23S kit (Qiagen).","Sample Treatment - Flasks were separated into 2 groups of 5 and each group was treated with either BVL3572S 27 µM or DMSO 0.8%. After 15 days at 37 °C, 1 mL of each culture was centrifuged (10,000 rpm, 2 min), washed with DPBS 1X supplemented with Tween 80 0.05% and diluted in fresh medium in order to obtain approximately 500,000 bacteria/mL in 25 mL. These cultures were incubated for 7 days at 37 °C up to an OD600 nm of approximately 0.6.","Nucleic Acid Extraction - Genomic DNA was extracted using the Quick-DNATM Fungal/Bacterial Miniprep Kit (Zymo Research).","Library Construction - Illumina sequencing libraires were prepared with the TruSeq RNA sample preparation kit version 2.0 rev. A (Illumina Inc.) with a unique index for each cDNA library.","Sample Treatment - Treatment was initiated by adding sub-MIC BVL3572S concentrations alone (0.85, 0.43, or 0.21 µM), with L-His 3 mM (0.43, 0.21, or 0.11 µM), with L-Ala 3 mM (1.70, 0.85, or 0.43 µM), or with 3 mM each of both L-His and L-Ala (13.63, 6.81, or 3.41 µM) to triplicate cultures. Antibiotic-free controls were prepared by adding DMSO 0.1% to cultures containing the corresponding amino acid. Cultures were outgrown for 14 days. On days 5 and 10 of outgrowth, medium was renewed by centrifuging the cultures and resuspending the pellets in medium before dilution to an OD600 nm of 0.1 in 7H9 containing kanamycin, ATc, and if applicable, BVL3572S and amino acid at the indicated concentrations. On day 14 of outgrowth, the impact of BVL3572S treatment on each culture was determined by measuring OD600 nm (the OD600 nm of untreated cultures was approximately 1). Cultures with partially drug inhibitory BVL3572S concentrations (growth reduction of 20 to 50%) and the corresponding controls were processed for genomic DNA extraction. Retained BVL3572S concentrations were 0.43 µM for the drug alone, 0.21 µM in the presence of L-His, 1.7 µM in the presence of L-Ala, and 6.81 µM in the presence of both amino acids.","Sample Treatment - Cultures were incubated with BVL3572S 20 µM or with DMSO 0.5% for 6 h at 37°C without agitation.","Growth Protocol - A pooled transposon mutant library of Mtb H37Rv, constructed by transduction with the MycoMar T7 phage (Payros et al. PLoS Pathogens 2021; DOI: 10.1371/journal.ppat.1010020) and containing more than 20,000 mutants, was pre-cultured in 5 mL of 7H9 medium supplemented with Tween 80 0.05%, albumin-dextrose-catalase 10% (ADC, BD Difco) and kanamycin (40 µg/mL) for 5 days at 37 °C. The culture was centrifuged (3,000 rpm, 10 min) and the pellet was washed with 15 mL of Dulbecco’s phosphate buffered saline (DPBS 1X, Gibco) with Tween 80 0.05%. Bacteria were resuspended in 25 mL of fresh medium without kanamycin and cultured for 2 days at 37 °C, until an OD600 of approximately 0.4 was reached. The culture was used to inoculate 10 flasks at an OD600 of 0.01 in 25 mL of medium. The flasks were incubated for 3 days at 37 °C until an OD600 nm of 0.2 was reached.","Library Construction - The sgRNA-encoding region was amplified from genomic DNA exactly as described in Li et al. Nature Microbiology 2022 (DOI: 10.1038/s41564-022-01130-y). Amplicons (~230 bp) were purified using NucleoMag magnetic beads (Macherey-Nagel 744970) using a double-sided size selection (bead:sample ratio of 0.6 followed by 1.0) and quantified using the Qubit dsDNA BR assay kit (ThermoFisher Scientific). Amplicon size and purity were visualized on a 1 % agarose gel and an Agilent 2100 bioanalyzer. For a more precise quantification, a qPCR assay was performed using the KAPA SYBR FAST qPCR kit (Roche KK4600).","Growth Protocol - 3 x 1mL Mtb CRISPRi library aliquots (RLC12, Addgene 163954) were thawed and inoculated in 10 mL of 7H9 containing kanamycin 20 µg/mL (OD600 nm = 0.1). After a 5-day incubation at 37 °C, cultures were centrifuged to get rid of clumps (100 x g, 2 min) and the supernatant was diluted to an OD600 nm of 0.1 in 40 mL of 7H9 containing kanamycin and anhydrotetracycline (ATc) at 100 ng/mL. Target pre-depletion was performed for 5 days after which, medium was renewed by centrifuging the cultures (3,500 x g, 5 min) and resuspending the pellet in 7H9 before dilution to an OD600 nm of 0.05 in 20 mL of kanamycin- and ATc-containing 7H9.","Sequencing - Individual PCR amplicons were multiplexed into 4 nM pools and sequenced on an Illumina Novaseq PE150 platform."],"figure_sub":["MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Resulting reads were mapped onto the Mtb H37Rv reference genome. For each sample, a table was generated containing the number of read for each TA insertion site. Data were processed using the TRANSIT pipeline. Insertion counts at each TA site were normalized using timed total reads (TTR) normalization. The resampling test module in TRANSIT was used to identify genes conditionally affecting fitness in the presence of BVL3572S treatment. Significant differences in read counts between untreated and treated conditions were assessed by comparison to a resampling distribution generated through random permutation of TA site counts within each genetic locus across all datasets. P values were calculated as the proportion of values from 10⁶ permutations that were more extreme than the observed result. To account for multiple comparisons, P values were adjusted using the Benjamini–Hochberg procedure to obtain false discovery rates.","Data Transformation - Raw reads were processed with Illumina quality control tools using default parameters. Sequences shorter than 50 bp, containing ambiguous bases (‘Ns’), and/or with a mean quality score lower than 30 were removed using PRINSEQ (http://prinseq.sourceforge.net/index.html). Next, rRNA-specific reads were excluded by aligning all reads on Mtb rRNA sequences using Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). Downstream RNA-seq analysis was performed using the SPARTA open-source software package with default settings (https://sparta.readthedocs.io/en/latest/).","Data Transformation - sgRNA sequences were extracted from single end reads using cutadapt between sequences [GTGATAGATATAATCTGGGA...GTTTTTGTAGCTCGAAAGAAG]. Extracted sequences shorter than 4 nucleotides were discarded after trimming. sgRNA sequences were mapped and quantified using MAGeCK count function. MAGeCK test function was used to determine sgRNA and gene level enrichment or depletion, using alphamedian L2FC method and Negative (control) sgRNA list for normalization (Li et al. Nature Microbiology 2022; DOI: 10.1038/s41564-022-01130-y)."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq X","Illumina NovaSeq 6000","NextSeq 500"],"study_type":["RNA-seq of coding RNA"],"species":["Mycobacterium tuberculosis H37Rv"],"pubmed_title":["Exploiting Vitamin B6 Dependency: BVL3572S Inhibits HisC and AlaA to Kill Mycobacterium tuberculosis"],"pubmed_authors":["Zainab Edoo, Astrid Lenne-Delmotte, Camille Grosse, Marie Devaere, Marion Michel, Guillaume Caron, Ernesto Anoz-Carbonell, Kamel Djaout, Rosangela Frita, Cyril Gaudin, Line Hofmann, Sophie Lecher, Véronique Megalizzi, Alessia Michelotti, David Rengel, Pauline Rouan, Stephanie Slupek, Lina Tawk, Rudy Antoine, Hanna Kulyk, Glenn Dale, Christophe Guilhot, Nicolas Willand, Guy Lippens, René Wintjens, Alain Baulard"],"additional_accession":[]},"is_claimable":false,"name":"RNA-seq and genome-wide CRISPRi and Tn-seq analyses of BVL3572S-treated Mycobacterium tuberculosis H37Rv","description":"(i) RNA-seq of M. tuberculosis H37Rv cultures grown in the presence of BVL3572S for 6 h compared to untreated controls.  (ii) Genome-wide M. tuberculosis H37Rv CRISPRi libraries were grown in the presence of ATc for 5 days prior to drug exposure. Cultures were treated with sub-MIC BVL3572S concentrations without amino acid or in the presence of L-His, L-Ala, or both. After 14 days outgrowth, genomic DNA were harvested from cultures for which BVL3572S concentrations were partially growth inhibitory and from untreated controls. Abundance of sgRNA was analyzed by deep sequencing. (iii) Genome-wide M. tuberculosis H37Rv transposon mutant librairies were treated with BVL3572S or untreated for 15 days followed by a 7-day antibiotic-free outgrowth. Genomic DNA were extracted, indexed, and sequenced.","dates":{"release":"2026-04-20T00:00:00Z","modification":"2026-04-20T10:09:09.533Z","creation":"2026-03-18T15:49:28.872Z"},"accession":"E-MTAB-16773","cross_references":{"ENA":["ERP190966"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0003969","EFO_0004184"]}}