organism: Mycobacterium tuberculosis H37Rv
description: Protocols: To isolate resistant mutants, exponentially growing cultures were concentrated, and about 1010 cells were plated onto 7H10 agar plates, containing different concentrations of a given drug, ranging from 4X- to 20X-MIC. Plates were incubated at 37 °C for 4 weeks. The resistant phenotype was validated by assessing the MIC at least twice on the isolated colonies. Resistant colonies were reinoculated in the presence of the compound (8X-MIC) and 10 mL of exponentially growing cultures were used for genomic DNA isolation. In short, each cell pellet was heat-inactivated at 80 ˚C for 1, washed and resuspended in 450 µL of bug lysis solution (25 mM Tris-HCl pH 7.9; 10 mM EDTA 50 mM glucose). 50 µL of lysozyme (10 mg/mL) were added and the cell suspension was incubated 37 ˚C overnight. Next, 100 µL of sodium dodecyl sulfate (10 %) and 50 µL of proteinase K (10 mg/mL) were added, mixed gently by inversion, and incubated at 55 ˚C for 30 min. Next, 200 µL of sodium chloride (5 M) were added and mixed gently by inversion, followed by 160 µL of preheated cetrimide saline solution (4 %), mixed and incubated at 65˚C for 10 min. Genomic DNA was extracted twice, adding equal volume (1 mL) of chloroform: isoamyl alcohol (24:1), mixing by inversion, and centrifuging for 5 min at 12000 RPM. The aqueous layer was transferred to a clean tube (800 µL) and 560 µL (0.7X volumes) of isopropanol were added at room temperature, mixed by inversion until DNA precipitation. DNA was collected by centrifugation for 10 min and the pellet was washed with 70 % ethanol, dried and resuspended in 100 µL of TE buffer. Libraries were built using a TruSeq DNA PCR-Free Low Throughput Library Prep Kit (Illumina, USA) following the manufacturer's protocol. Quality control was performed on an Agilent BioAnalyzer.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-12-31
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-12-31
attributes.tag: External Id
attributes.value: SAMEA111479730
attributes.tag: INSDC center alias
attributes.value: 1. Institut Pasteur, Universite Paris Cite, Plate-forme Technologique Biomics, F-75015 Paris, France 2. Institut Pasteur, Universite Paris Cite, Bioinformatics and Biostatistics Hub, F-75015 Paris, France
attributes.tag: INSDC center name
attributes.value: 1. Institut Pasteur, Universite Paris Cite, Plate-forme Technologique Biomics, F-75015 Paris, France 2. Institut Pasteur, Universite Paris Cite, Bioinformatics and Biostatistics Hub, F-75015 Paris, France
attributes.tag: INSDC first public
attributes.value: 2022-12-31T00:58:39Z
attributes.tag: INSDC last update
attributes.value: 2022-12-31T00:58:39Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12307:36009_H1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: TMC 102
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: logarithmic phase culture
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: sample name
attributes.value: E-MTAB-12307:36009_H1