7_oxB_in
Mycolicibacterium smegmatis MC2 155
Protocols: Mycobacterium smegmatis mc2-155 SMR5 wild-type cells were grown as shaking culture in Middlebrook 7H9 (supplemented with 0.2% v/v glycerol, 0.05% w/v Tween-80, and 0.2% w/v glucose) to an OD600 between 0.5 to 0.7 at 37 °C. The culture was split into 50 ml aliquots and cells were collected by centrifugation (2000 g, 10 min, 25 °C), resuspended in the same volume of fresh medium. DNA damage stress (100 ng/ml MMC) or oxidative stress (5 mM H2O2) was applied and cultures were incubated further at 37 °C with shaking. After 4 h, cultures were equilibrated to room temperature for 20 min before crosslinking by addition of formaldehyde (Thermo Scientific #28908; 1% w/v final concentration) for 20 min at room temperature with agitation. The crosslink reaction was quenched by addition of glycine to 250 mM and incubation for 5 min at room temperature. Crosslinked cells were collected by centrifugation (2000 g, 10 min, 4 °C) and washed twice in 40 ml to 50 ml cold PBS (4 °C). Samples were immediately processed with the nucleic acid extraction protocol. Reads were quality trimmed using Trimmomatic v0.35 (LEADING=3, SLIDINGWINDOW=4:15, MINLEN=30). Pellets were resuspended in 800 μl lysis buffer (20 mM HEPES-KOH pH 7.9/RT, 50 mM KCl, 0.1% v/v Triton X-100, 0.5 mM DTT, 1 mM EDTA, 1x c0mplete EDTA-free protease inhibitor cocktail (Roche), 1 mM PMSF), transferred to 2 ml screw cap tubes containing 500 mg to 700 mg 0.15 mm zirconium oxide beads (NextAdvance #ZrOB015). Cell lysis was performed by bead beating (3 x 30 s, 5 m/s, 5 min pause on ice in between, MP FastPrep-24). Beads and crude cell material were sedimented by mild centrifugation (2 x 100 g, 2 min, 4 °C, inverting tubes once in between) and the supernatant was transferred to new tubes. DNA was sheared using a Covaris E220 instrument (microTUBES AFA fibre, 130 μl; 10% duty cycle, 175 W PIP, 200 cbp, 400 s to 430 s duration) to a fragment length between 200 bp to 300 bp. Insoluble material was removed by centrifugation (11000 g, 10 min, 4 °C) and the supernatant was transferred to new tubes. 80 μl supernatant were set aside as “input” sample and supplemented with TrisHCl pH 8.8 (180 mM final) and EDTA (2.5 mM final). Buffer conditions in the cleared lysate used for immunoprecipitation were adjusted by addition of TrisCl pH 8/RT (10 mM final) and NaCl (150 mM final). Crosslinked PafBC-DNA complexes were captured by addition of 8 μg anti-PafBC antibody and incubation over night at 4 °C. Captured complexes were precipitated by addition of 40 μl Protein A Magnetic Beads (NEB #S1425S) previously washed in IPP150 buffer (10 mM TrisHCl pH 8/RT, 150 mM NaCl, 0.1% v/v Triton X-100) followed by incubation at room temperature for 2 h with agitation. Beads were washed five times in 900 μl IPP150 buffer and two times in 900 μl TE buffer (50 mM TrisHCl pH 8/RT, 10 mM EDTA). To elute bound complexes, beads were resuspended in 150 μl or 100 μl TE buffer containing 1% w/v SDS and incubated for 15 min at 65 °C. A second elution was performed with 100 μl or 50 μl for 5 min at 65 °C and the eluates of both steps were pooled (“enriched” sample). Enriched and input samples were treated with RNase A (0.2 mg/ml for 30 min at room temperature) and Proteinase K (0.4 mg/ml for 1 h at 55 °C) followed by reversal of the formaldehyde crosslinks at 65 °C over night. DNA fragments were purified from the input and enriched samples using the ChIP DNA Clean & Concentrator Kit (Zymo #D5201; elution in 16 μl 5 mM TrisHCl pH 8.5, 0.1 mM EDTA). DNA concentrations were determined using the Quant-iT PicoGreen dsDNA Assay Kit (ThermoScientific #P7589). Sequencing libraries were prepared with 100 ng DNA for input samples and 10 ng for enriched samples using the Ion Plus Fragment Library Kit (ThermoScientific #4471252) together with an Ion Xpress Barcode Adapters Kit (ThermoScientific) following the manufacturer's protocol. Size-selection during library preparation was performed using AMPure XP beads (Agencourt) selecting fragments from 160 bp to 350 bp length.
ENA first public
2018-06-17
ENA last update
2018-02-14
External Id
SAMEA104590939
INSDC center alias
ETH Zurich Institute of Molecular Biology and Biophysics
INSDC center name
ETH Zurich Institute of Molecular Biology and Biophysics
INSDC first public
2018-06-17T17:02:46Z
INSDC last update
2018-02-14T14:45:07Z
INSDC status
public
Submitter Id
E-MTAB-6503:7_oxB_in
broker name
ArrayExpress
genotype
wild type genotype
growth condition
Middlebrook 7H9
sample name
E-MTAB-6503:7_oxB_in