Neurospora cultures were crosslinked with 1% FA for 10 min. FA was quenched 5 min with 125 mM glycine. ∼ 400 μl ground mycelial powder was resuspended and digested in 600 μl MNase digestion buffer for 1 h at 25°C with 15 U of MNase (Sigma-N3755-500) (In addition an independent sample from a 1 min light-induced culture was digested with 5 U MNase). The reactions were stopped by adding final 5 mM EDTA pH 8.0. Then 400 μl ChIP lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1% Triton-X, 0.1% SDS and 0.1% NaDOC) was added and samples were centrifuged at 4°C, 15000 g. The rest of the ChIP protocol was performed as described [19 ]. DNA for ChIP-seq was pooled from two-independent experiments.
Mnase
Manufactured by Merck Group
Sourced in United Kingdom
MNase is a laboratory equipment product from Merck Group. It is an enzyme used for digesting chromatin. MNase cleaves DNA between nucleosomes, allowing for the isolation and purification of mononucleosomes.
Automatically generated - may contain errors
Lab products found in correlation
Dna purification kit, by Tiangen Biotech (3 mentions)
H3k4me1, by Abcam (3 mentions)
Ab8580, by Abcam (3 mentions)
H3k27ac, by Abcam (3 mentions)
Novaseq 6000, by Illumina (3 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Proteinase k, by Merck Group (2 mentions)
β agarase, by Merck Group (2 mentions)
Ab8895, by Abcam (2 mentions)
Ab472, by Abcam (2 mentions)
Ku55933, by R&D Systems (2 mentions)
Gfp 11 814 460 001, by Roche (2 mentions)
Cpd tdm 2, by MBL Life Science (2 mentions)
Dna modifying enzymes, by Roche (2 mentions)
Nu7441, by R&D Systems (2 mentions)
Rnaseh2a nbp1 76981, by Novus Biologicals (2 mentions)
Snrnp40 sab2701506 and srsf2 sc35 clone sc 35, by Merck Group (2 mentions)
Snrpa u1a 3f9 1f7, by Abcepta (2 mentions)
Phospho chk2 thr68 2661, by Cell Signaling Technology (2 mentions)
Odyssey compatible irdye680 and irdye800 conjugated secondary antibodies, by LI COR (2 mentions)
65 protocols using mnase
1
Chromatin Immunoprecipitation in Neurospora
2
ChIP-seq of H3K27me3 in Sperm
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ChIP-seq in sperm was performed using a native ChIP protocol according to Hisano et al. (2013) (link). Briefly, sperm were resuspended in 1 mL cold PBS. 50 ul of 1M DTT was added and samples were incubated for 2 hr at room temperature. 120 ul 1M N-ethylmaleimide (#P4557, Sigma Aldrich) was added and the sample was incubated for another 20 min at room temperature. An aliquot was removed as a pre-MNase control, the sample was digested with 10 units of MNase (#10107921001, Sigma Aldrich) for 5 min at 37C, and 2 ul 0.5M EDTA was added to stop the digest. The chromatin solution was precleared for 1 hr with pre-blocked Protein G Dynabeads, then removed from the beads. 100 ul was set aside as a pre-ChIP control, and the remainder of the sample was incubated with 5 ug anti-H3K27me3 (ab6002, Abcam) overnight at 4C. The following day, chromatin samples were incubated for 8 hr with pre-blocked beads, then washed, eluted from the beads, and treated with RNAse A and proteinase K. DNA was purified using a Zymo ChIP Clean and Concentrator kit. Two biological replicates were prepared for each of the control and Kdm6a cKO genotypes. For each replicate, sperm from five males was pooled in a single ChIP experiment in order to recover enough histones for robust ChIP.
3
ChIP-Seq Profiling of Chromatin Modifications
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Chromatin immunoprecipitation (ChIP) assays were performed as previously described [24 (link)]. Soluble chromatin was prepared using MNase digestion [50 mM Tris-HCl, pH 7.5, 4 mM MgCl2, 1 mM CaCl2, 0.32 mM sucrose, 2 mM NaF, 0.2 mM NaVan] with 2 units of MNase (Sigma, UK)/180 μg chromatin, then immunoprecipitated with H300 GR antibody (Santa Cruz; sc-8992), RNA Polymerase II (phosphoserine-5) antibody (ACTIVE MOTIF; 39233) or rabbit non-immune serum (Santa Cruz; sc-2027). For sequencing, libraries were prepared from two replicates/condition. RT-qPCR was performed using SYBR reagents (Applied Biosystems) and primers described in [24 (link)] (Supplementary Table S6 ).
4
Micrococcal Nuclease Chromatin Mapping
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MNase assays were performed essentially as in Torigoe et al. (2013) (link) and Alexiadis (2002) (link). MNase (Sigma-Aldrich, St. Louis) was resuspended at 200 units/mL in water and then disluted at 1:100, 1:500, or 1:1000 in a solution of 1X MNase reaction buffer (50 mM Tris-HCl pH7.9 and 5 mM CaCl2 dihydrate), 0.1 mg/mL insulin, and 10% glycerol. Chromatinized plasmid assemblies in 1X MNase buffer were added to each MNase dilution. As a control, an equal amount of unchromatinized super-coiled plasmid was added to the lowest MNase dilution. Each reaction was incubated for 11 min at room temperature, then stopped in a solution with final concentrations of 20 mM EGTA, 200 mM NaCl, 1% SDS, 20 mg/mL GlycoBlue (Thermo Fisher Scientific, Waltham, MA), and 13.4 mg/mL Proteinase K (Roche, Basel, Switzerland) and incubated at 37°C for 30 min. Reactions wer Phenol/Chloroform/Isoamyl Alcohol extracted, ethanol precipitated, and run on a 1.3% agarose gel in 0.5X TBE. 3 μg of the 123 bp DNA ladder (Thermo Fisher Scientific, Waltham, MA) was used as a size standard.
5
Chromatin Preparation and MNase Digestion
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Samples were thawed slowly on ice and adjusted to a final volume of 1 ml with supplemented S1 buffer and Dounce homogenized. Lysate was centrifuged at 2000xg (4 °C) and lysed in supplemented (2 mM NaF and 0.2 mM sodium orthovanadate and 1X cOmplete protease inhibitor) sodium dodecyl sulphate (SDS) lysis buffer (2% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1)). Soluble chromatin was prepared according to a protocol developed in house71 (link),72 (link) using MNase digestion [50 mM Tris-HCl, pH 7.5, 4 mM MgCl2, 1 mM CaCl2, 0.32 mM sucrose, 2 mM NaF, 0.2 mM NaVan] with 2 units of MNase (Sigma, UK)/180 μg chromatin.
6
Mononucleosomal DNA Sequencing by MNase Digestion
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Samples containing 105 macronuclei were incubated in the digestion buffer (0.25 M sucrose, 10 mM MgCl2,10 mM Tris at pH 7.4, 1 mM CaCl2) with increasing amounts (0, 0.5, 1, 2, 5, 7.5, 10 U) of MNase (Sigma-Aldrich) for 10 min at 30°C. Reactions were stopped by the addition of three volumes of 0.5 M EDTA (pH 9.0), 1% N-lauroylsarcosine (Sigma-Aldrich), 1% SDS, 1 mg/mL Proteinase K (Merck) and incubated overnight at 55°C. DNA from each sample was gently extracted once with phenol and dialyzed twice against TE (10 mM Tris-HC1, 1 mM EDTA at pH 8.0) containing 25% ethanol and once against TE. Samples were then treated with RNase A, and DNA was quantified with a NanoDrop spectrophotometer (Thermo Fisher Scientific) and separated on a 1.2% agarose gel. The reactions containing mostly mononucleosomal DNA fragments (see Fig. 1 ) were selected, and mononucleosomal DNA fragments were purified from 3% low melting-temperature agarose gels and treated with β-agarase (Sigma-Aldrich) for sequencing.
7
Chromatin Immunoprecipitation Assay Protocol
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ChIP assay was performed as previously described [40 (link)]. Briefly, approximately 1 × 107 cells were fixed with 1 % formaldehyde and quenched by glycine. The cells were washed three times with PBS and then harvested in ChIP lysis buffer (50 mM Tris-HCl, pH 7.6, 1 mM CaCl2, 0.2 % Triton X-100). DNA was digested to 150–300 bp by MNase (Sigma) before extensive centrifugation. Four volumes of ChIP dilution buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 % Triton X-100, 0.1 % SDS) was added to the supernatant. The resulted lysate was then incubated with protein G beads and antibodies at 4 °C over night. The beads were washed five times and DNA was eluted by Chip elution buffer (0.1 M NaHCO3, 1 % SDS, 20 μg/ml proteinase K). The elution was incubated at 65 °C over night and DNA was extracted with DNA purification kit (TIANGEN). The purified DNA was assayed by quantitative PCR with Biorad MyIQ. Assays were repeated at least three times. Data shown were average values ± SD of representative experiments. The sequences of primers are in Additional file 2 : Table S16.
8
Micrococcal Nuclease Digestion of Chromatin
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1 × 107 U2OS cells were collected and washed with cold PBS, and incubated in lysis buffer (300 mM HEPES pH7.5, 60 mM KCl, 300 mM sucrose, 5 mM K2HPO4, 5 mM MgCl2, 2 mM EDTA, and 0.5 % Triton X-100) for 5 min on ice. The cells were then broken with 7 gentle strokes in a type B Dounce homogenizer. Nuclei were pelleted by centrifuging at 120 × g for 10 min. The pelleted nuclei were washed with 1 ml MNase digestion buffer (10 mM Tris pH7.5, 15 mM NaCl, 1 mM CaCl2, 60 mM KCl, and 0.2 mM phenyl methyl sulfonate fluoride), resuspended in 1 ml MNase digestion buffer, and digested with 0.2 U/ml (Sigma units) of MNase at 37 °C for various lengths of time. Digestions were stopped with 10 mM EDTA followed by incubation with 0.1 mg/ml proteinase K at 37 °C for 5 min. The resulting DNA samples were isolated using phenol extraction and ethanol precipitation, and resolved on 1 % agarose gel [61 ].
9
MNase Chromatin Fractionation and DNA Analysis
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Chromatin fractions acquired as described above were resuspended in MNase buffer (10 mM Tris, 10 mM KCl, and 1 mM CaCl2), and MNase (Sigma-Aldrich) was added. After incubation at 37 °C for 5 min, the reaction was stopped with EDTA (1 mM, final concentration). Then RNase A and protein K were added into the mixture for 6 h at 65 °C. DNA was column-purified (QIAquick Spin Kit; Qiagen, Valencia, CA) and analyzed by 2% agarose gel electrophoresis.
10
Neutrophil Extracellular Trap (NET) Formation
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A total of 6 × 106/1 mL freshly isolated neutrophils were primed with 25 ng/mL GM‐CSF in DMEM for 30 minutes in 24‐well plates (Costar) and then, incubated in duplicates for 3 hours in medium, with 25 nM PMA, 5 µM ionomycin, or alum at a concentration of 100 µg/mL at 37°C and 5% CO2. Twenty micro liter of a solution of 500 mU/mL MNase (Sigma‐Aldrich) in distilled water were added to the cultures and incubated for 20 minutes at 37°C. Thereafter, the culture supernatants were harvested and remaining cells were removed by centrifugation at 400 g for 5 minutes. These supernatants were collected and termed “NETs.”
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