organism: Mycobacterium tuberculosis H37Rv
description: Protocols: Primary cultures were grown up to OD600nm 0.5/0.6 and diluted to OD600nm 0.025 using pre-warmed Middlebrook 7H9 broth. Secondary cultures were grown up to OD600nm 0.25 and drugs were added at a concentration equal to 10 fold the minimal inhibitory concentration for 6 hours. Cells were collected by centrifugation for 10 minutes and frozen at -80 °C, before RNA isolation. RNA was extracted by the hot phenol method. In detail, bacterial cell pellets were processed in 400 µL of hot lysis buffer (50 mM Tris pH 8.0, 1 mM EDTA, 1% SDS) and then mixed in another screw cap tube containing 400 µL of hot phenol (pH 6.6) and 400 µL of acidified glass beads (425-600-µm diameter). Cells were incubated at 65 °C for 15 minutes with intermittent vortexing every 5 minutes. Samples were centrifuged at 10,000 rpm for 15 minutes at 4 °C. The aqueous phase was separated into another fresh screw-capped microcentrifuge tube, an equal amount of Trizol and 100 µl of chloroform were added and mixed gently by inversion. The samples were again centrifuged at 10,000 rpm for 10 minutes at 4 °C, the aqueous phase separated into a fresh tube, and the RNA was precipitated with 0.5 M LiCl and three volumes of cold 100% ethanol. Samples were mixed gently by inversion, and then precipitated for 2 hours at -20 °C. Samples were centrifuged at 13,500 rpm for 35 minutes at 4 °C and RNA was washed with 80% ethanol. RNA samples were air-dried for 30 minutes and resuspended in 20 µL of nuclease-free water. Finally, DNA was depleted by TURBO DNase treatment (Ambion), according to the manufacturer's instructions. The RNA integrity index was examined on the Agilent Bioanalyzer RNA 6000 Pico kit to confirm the quality and integrity of the RNA for RNAseq purposes. For RTq-PCR, the concentration was measured via NanoDrop 2000/2000c spectrophotometer. rRNA was depleted using the QIAseq FastSelect 5S/16S/23S kit for bacteria (Qiagen),Ribosoma vailableaccording to manufacturer's recommendations. The libraries were prepared using the TruSeq Stranded mRNA Library Preparation kit (Illumina) according to manufacturer's protocol https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_truseq/truseq-stranded-mrna-workflow/truseq-stranded-mrna-workflow-checklist-1000000040600-00.pdf. The quality, integrity and amount of the libraries were assayed with the Agilent 2100 Bioanalyzer and Qubit fluorimeter.
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: SAMEA111479754
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-12306:34503_2
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-12306:34503_2