{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["Jarmila Hnilicova"],"instrument_platform":["NextSeq 550"],"study_type":["ChIP-seq"],"organism":["Mycolicibacterium smegmatis MC2 155"],"species":["Mycolicibacterium smegmatis MC2 155"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-12348"],"description":["CsrL (MSMEG_5890) associates with CarD and RNA polymerase in mycobacteria. For ChIP-seq, we used Mycobacterium smegmatis strain with CrsL (LK3051), where an additional copy of CrsL under anhydrotetracycline inducible promoter was stably inserted into M. smegmatis genome. CrsL-FLAG was expressed in exponential phase and ChIP performed at mid-exponential phase of growth."],"repository":["biostudies-arrayexpress"],"sample_protocol":["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.","Growth Protocol - M. smegmatis mc2 155 strain LK3051 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.","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."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","MAGE-TAB Files"],"pubmed_authors":["Mahmoud Shoman","Jarmila Hnilicova","Martin Převorovský"],"additional_accession":[]},"is_claimable":false,"name":"ChIP-seq identification of CrsL (MSMEG_5890) binding sites in Mycobacterium smegmatis","description":"CsrL (MSMEG_5890) associates with CarD and RNA polymerase in mycobacteria. For ChIP-seq, we used Mycobacterium smegmatis strain with CrsL (LK3051), where an additional copy of CrsL under anhydrotetracycline inducible promoter was stably inserted into M. smegmatis genome. CrsL-FLAG was expressed in exponential phase and ChIP performed at mid-exponential phase of growth.","dates":{"release":"2025-11-01T00:00:00Z","modification":"2025-11-01T02:01:47.319Z","creation":"2022-10-31T14:51:06.325Z"},"accession":"E-MTAB-12348","cross_references":{"ENA":["ERP142171"],"Biostudies":["E-MTAB-12349","E-MTAB-7004","E-MTAB-11828","E-MTAB-12350","E-MTAB-12351"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0002692","EFO_0005518","EFO_0004184"]}}