2025-10-30
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA111532403
External Id
Department of Microbial Genetics and Gene Expression, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic
INSDC center name
2022-10-31T14:37:44Z
INSDC last update
public
INSDC status
ERS13642445
SRA accession
E-MTAB-12348:ChIP_CrsL-FLAG_3
Submitter Id
ArrayExpress
broker name
Protocols: 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. 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. 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. 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.
description
CrsL-FLAG
genotype
Middlebrook 7H9 medium
growth condition
not applicable
isolate
Mycolicibacterium smegmatis MC2 155
organism
Mycolicibacterium smegmatis MC2 155
organism
whole organism
organism part
Mycolicibacterium smegmatis MC2 155
scientific_name
LK3051
strain
ChIP_CrsL-FLAG_3
title