biostudies-arrayexpressMycolicibacterium smegmatis MC2 155https://www.ebi.ac.uk/biostudies/studies/E-MTAB-11828CarD is essential transcription factors in mycobacteria. For ChIP-seq, we used Mycobacterium smegmatis strains with CarD-FLAG (LK2539), where an additional copy of CarD under anhydrotetracycline inducible promoter was stably inserted into the genome. CarD-FLAG was expressed in exponential phase and ChIP performed at mid-exponential phase of growth.biostudies-arrayexpressGrowth Protocol - M. smegmatis mc2 155 strain LK2539 was grown at 37°C in Middlebrook 7H9 medium with 0.2% glycerol and 0.05% Tween 80. Cells were inoculated to OD600 0.1 in 100 ml of 7H9/Tween/glycerol, anhydrotetracycline was added after 3 hours of growth (10 ng/ml final concentration). After additional 3 hours (totals 6 hours of growth) cells were harvested.Nucleic Acid Extraction - Bacterial pellet was resuspended in 3 ml of ice cold RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% dexycholate, 0.1% SDS, 50 mM Tris-Cl pH 8.0, 5 mM EDTA) with protease inhibitors cocktail (Sigma-Aldrich, 5 ul/1 ml) and 0.1 M PMSF (5 ul/1 ml) and sonicated 18x10 seconds with 1 min pauses on ice between cycles. Lysate was centrifuged 9000 rpm at 4°C for 15 min and pellet discarded. 2 mg of supernatant was incubated with 20 ul of M2 anti-FLAG resin (Sigma-Aldrich) overnight at 4°C. M2 resin was rinsed twice with RIPA, four times with 100 mM Tris-HCl, pH 8.5, 500 mM LiCL, 1% Triton X-100, 1% deoxycholic acid, twice again with RIPA and twice with TE. Protein-DNA complexes were eluted with 1% SDS for 10 minutes at 65°C, decrosslinked in the presence of 200 mM NaCl for 5 h at 65°C, treated with 100 ug/ml RNAse A for 1 hour at 37°C and 20 µg proteinase K for 30 min at 45°C. DNA was purified with Qiagen PCR purification kit and eluted into 100 ul EB.Sequencing - Pooled barcoded libraries (one biological triplicate) were sequenced in single lane with Illumina NextSeq® 500/550 High Output Kit v2 in 75 bp single end regime at Institute of Molecular Genetics AS CR, Prague, Czech Republic.Library Construction - 40 ul of immunoprecipitated DNA sample or 10 ng of DNA input were used for library construction according to the NEXTflex™ ChIP-Seq Kit manual including the Size-Selection Cleanup step B2.Sample Collection - 100 ml of bacterial culture was crosslinked with 1% formaldehyde (final conc.), formaldehyde was added directly to the medium) for 30 minutes at 37°C in the shaker. Formaldehyde was quenched by glycine (0.125 M final conc.) added for 5 minutes at 37°C in the shaker. Bacteria were centrifuged for 5 min at 9000 rpm, 4°C, pellet washed with 10 ml of 1x PBS, bacteria centrifuged again and pellet immediately frozen at -80°C.MINSEQE ScoreAssays and DataorganisationMAGE-TAB FilesMetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 550ChIP-seqMycolicibacterium smegmatis MC2 155E-MTAB-7004Jarmila HnilicovaViola Vaňková HausnerováMartin PřevorovskýfalseChIP-seq identification of CarD (MSMEG_6077) binding sites in Mycobacterium smegmatisCarD is essential transcription factors in mycobacteria. For ChIP-seq, we used Mycobacterium smegmatis strains with CarD-FLAG (LK2539), where an additional copy of CarD under anhydrotetracycline inducible promoter was stably inserted into the genome. CarD-FLAG was expressed in exponential phase and ChIP performed at mid-exponential phase of growth.2025-11-01T00:00:00Z2025-11-01T02:01:47.322Z2022-06-15T17:30:47.126ZE-MTAB-11828ERP138375E-MTAB-7004EFO_0002944EFO_0004170EFO_0003789EFO_0002692EFO_0005518EFO_0004184