To clarify sRNAs inside or outside EVs, P100 vesicles were treated with micrococcal (MNase) and proteinase K. For MNase treatment, P100 vesicles was treated with 10 U of MNase (Thermo Fisher) for 15 minutes at 37°C with or without Triton X-100. For proteinase K plus MNase treatment, P100 vesicles was treated with 20 μg/ml proteinase K (Invitrogen) for 1 hour at 37°C with or without Triton X-100. The proteinase activity was inhibited by adding 5 mM phenylmethylsulfonyl fluoride for 10 minutes at room temperature. The sample was then treated with 10 U of MNase for 15 minutes at 37°C. For Triton-X-100 treatment, P100 vesicles were incubated with 1% (v/v) Triton-X-100 on ice for 30 minutes before proteinase and nuclease treatments. Immediately after proteinase and nuclease treatments, RNA was extracted for further detection of specific sRNAs. sRNA RT-PCR was performed as previously described (Cai et al., 2018 (link)).
Mnase
Manufactured by Thermo Fisher Scientific
Sourced in United States
MNase is a lab equipment product from Thermo Fisher Scientific. It is an enzyme that specifically cleaves DNA between nucleosomes, allowing the study of chromatin structure and dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase k, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Minelute pcr purification kit, by Qiagen (3 mentions)
Anti h3k27me3 chip, by Cell Signaling Technology (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Mnase, by Roche (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Nuclei isolation buffer, by Abcam (2 mentions)
Rnase inhibitor, by Takara Bio (2 mentions)
Sarkosyl, by Merck Group (2 mentions)
Sodium deoxycholate, by Merck Group (2 mentions)
Fusion 200, by Chemyx (2 mentions)
Mnase, by New England Biolabs (2 mentions)
Rnase a, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Freeze n squeeze column, by Bio-Rad (1 mentions)
Dnazol, by Molecular Research Center (1 mentions)
Picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
49 protocols using mnase
1
Distinguishing sRNA Localization in EVs
2
Nucleosome Mapping by MNase Digestion
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A total of 1 × 107 cells were harvested and cell pellets were resuspended in hypotonic lysis buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl2 and 0.5% NP-40) for 5 min on ice. Cell nuclei were pelleted (5 min, 120 × g), washed once in digestion buffer (50 mM Tris pH 8, 5 mM CaCl2) and resuspended in digestion buffer. MNase (cat# EN0181, Thermo Fisher Scientific, Waltham, MA, USA) was diluted in MNase storage buffer (20 mM Tris pH 7.6, 50 mM NaCl, 50% glycerol), and 0 U, 0.1 U, 0.5 U, 0.75 U, 1 U and 3 U were added to the nuclei in a final volume of 100 μl. Reactions were incubated for 5 min at 37°C, stopped by the addition of stopping buffer (20 mM EDTA, 0.4% SDS, 0.5 mg/ml proteinase K) and incubated at 50°C overnight. DNA was recovered by adding 0.1 volumes of 3 M NaCl and 2 volumes of 100% ethanol and storage at –20° for 1 h. After centrifugation (15 min, 15 000 × g) and washing with 70% ethanol, the DNA pellet was air-dried and resuspended in 50 μl ddH2O. Equal amounts of DNA were loaded on a 1.5% agarose gel and run for 45 min at 100 V. Gels were stained with EtBr in 1× TAE as described above and imaged using a AI600 imager (Supplementary Table S6 ). Integrated intensities of monomeric DNA and total DNA in each lane were measured with ImageJ and plotted as a DNAmononmer/DNAtotal ratio.
3
Nuclei Isolation and MNase Digestion
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After harvesting the cells (1x106 cells per sample/treatment), nuclei were isolated using a hypotonic buffer (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.1% NP-40). The isolated nuclei were digested at 37°C for different periods of time with 0.5 U MNase (Thermo Scientific) in 650 μl of MNase digestion buffer (50 mM Tris–HCl pH 8.0, 5 mM CaCl2). Aliquots of 100 μl were taken every 8 min and MNase activity was inactivated by adding 0.5 M EDTA pH 8.0. DNA isolation was performed by adding SDS to a final concentration of 1% and incubating with RNase A (200 μg/ml) at 37°C for 30 min. Next, Proteinase K (400 μg/ml) was added and the sample was incubated at 55°C for 2 h. Genomic DNA was purified by phenol-chloroform extraction followed by ethanol/acetate precipitation. Similar amounts of the partially digested DNA were loaded on a 2.0% agarose gel and separated by electrophoresis.
4
Micrococcus Nuclease Digestion Analysis
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For Micrococcus nuclease assays, whole cell lysates were layered onto a 1.2 M sucrose cushion (1.2 M sucrose in 60 mM, KCl 15 mM NaCl, 5 mM MgCl2, 0.1 mM ethylene glycol tetraacetic acid (EGTA), 15 mM Tris–HCl (pH 7.5), 0.5 mM DTT, 0.1 mM phenylmethanesulfonylfluoride (PMSF), and 3.6 ng/mL aprotinin) and centrifuged as described elsewhere [41 (link)]. Nuclei were collected and homogenized in Micrococcus nuclease (MNase) digestion buffer (0.32 M sucrose, 50 mM Tris–HCl (pH 7.5), 4 mM MgCl2, 1 mM CaCl2, and 0.1 mM PMSF). MNase digest time courses of each 1 × 106 cells were performed for 0, 1, 2, 4, 8, 16 or 20 minutes with 0.15 U/μL MNase (Thermo Fisher Scientific, Waltham, MA, USA). The reactions were stopped by directly mixing 50 μL phenol:chloroform:isoamylalcohol with each sample followed by DNA extraction. From the aqueous phase DNA was precipitated with isopropanol, resolved in water and DNA fragments were analyzed on an Agilent Bioanalyzer 2100 using a DNA chip.
5
Chromatin Immunoprecipitation and RNA Sequencing
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ChIP-seq were performed as described in previously [4 (link),6 (link)]. Briefly, cells were dual cross-linked with 2 mM ethylene glycol bis (succinimidyl succinate) (EGS) and disuccinimidyl glutarate (DSG) and 1% formaldehyde. Chromatin was isolated and then fragmented with 60 units MNase (New England Biolabs, M0247S) at 37 0C for 10 minutes. For β-catenin ChIP-seq, cells were first fractionated by Subcellular Protein Fractionation Kit for Cultured Cells (ThermoFisher, Cat# 78840) to obtain a nuclear fraction that was diluted 1:5 with MNase digestion buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1% Triton X-100, 1 mM CaCl2, 1 mM DTT), and then digested with MNase as above. Antibodies used in the ChIP-seq experiments were against β-catenin (Cell Signaling, Cat#9581), WDR77 (SCBT, Cat# sc-100899), and MafB (SCBT, Cat# sc-10022).
For RNA-seq experiments, mRNeasy Mini Kit (Qiagen, No. 217004) was used for RNA extraction. 0.4 μg total RNA was used for RNA-seq library construction by a TruSeq RNA Library Prep Kit V2 (Illumina, RS-122-2001). Both ChIP-seq and RNA-seq libraries were sequenced at the Bauer Core Facility, Harvard.
For RNA-seq experiments, mRNeasy Mini Kit (Qiagen, No. 217004) was used for RNA extraction. 0.4 μg total RNA was used for RNA-seq library construction by a TruSeq RNA Library Prep Kit V2 (Illumina, RS-122-2001). Both ChIP-seq and RNA-seq libraries were sequenced at the Bauer Core Facility, Harvard.
6
MNase Digestion and Quantification
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DT40 cells were washed with ice‐cold PBS and incubated for 5 min in ice‐cold NP‐40 cell lysis buffer (10 mM Tris–Cl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P‐40, 0.15 mM spermine, 0.5 mM spermidine). Cells were washed in MNase digestion buffer (10 mM Tris–Cl pH 7.4, 15 mM NaCl, 60 mM KCl, 0.15 mM spermine and 0.5 mM spermidine) and then resuspended in MNase digestion buffer supplemented with 1 mM CaCl2 to a final density of 100 × 106 cells/ml. Two hundred microlitres of the cellular dilution was incubated with or without (total input) 80 U of MNase (Thermo Scientific) for 30 min at 37°C, after which the reaction was stopped by adding 160 μl of MNase digestion buffer and 66 μl of stop buffer (60.6 mM EDTA, 6 mM EGTA, 6% SDS and 2.27 mg/ml of proteinase K). The samples were phenol–chloroform‐extracted, treated with 20 μg of RNase for 2 h at 37°C, phenol–chloroform‐extracted again and washed by ethanol–acetate precipitation. The MNase‐resistant DNA was quantified by qPCR using the primer pairs detailed in Table EV2 .
7
Reconstituted Chromatin Micrococcal Nuclease Assay
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As DNA template for reconstituted chromatin samples, a ~0.5 kb region from the M. jannaschii RNAP operon was PCR-amplified using primers FW1522 and FW1523 (Supplementary Table 1 ). Overall, 20 µl samples contained 1 µg PCR product and 0.73 µg A3, 1.14 µg MJ1647 or 0.79 µg MJ1647ΔTM in N200 buffer. Samples were incubated at 37 °C for 15 min before the addition of 80 µl 1.25× MNase digestion buffer (25 mM Tris/HCl pH 8.0, 31.25 mM NaCl, 6.25 mM CaCl2) containing 1, 3 or 10 units MNase (Thermo Fisher). Samples were further incubated at 37 °C for 5 min before the addition of 10 µl stop buffer (2% SDS, 0.1 M EDTA pH 8.0). After Phenol:Chloroform purification and ethanol precipitation, the samples were resolved on 3% agarose gels post-stained with ethidium bromide.
8
Chromatin Fractionation from Nuclei
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Nuclei from 120 × 106 cells for each treatment condition were prepared as described before (Gilbert et al., 2003 (link)) with an NP-40 concentration of 0.5% in buffer B. For MNase digest, the nuclei concentration was adjusted to 20 A260 in nuclei buffer R (85 mM KCL, 5.5% sucrose, 10 mM Tris, pH 7.5, 1 mM CaCl2, 1 mM MgCl2, and 250 µM PMSF) and digested with 8 U MNase (Thermo Fisher Scientific) for 8 min at RT. Digestion was stopped by addition of EDTA to 10 mM, the sample was spun down, and chromatin was released overnight at 4°C in TEEP20 (10 mM Tris, pH 8, 1 mM EDTA, 1 mM EGTA, 250 µM PMSF, and 20 mM NaCl) containing 300 ng/ml l -α-lysophosphatidylcholine (Sigma-Aldrich). Nuclear debris was removed through centrifugation, and the soluble chromatin was divided into two samples (one brought to 650 mM NaCl) before fractionation on sucrose step gradients.
9
Chromatin Fractionation and MNase Digestion
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Briefly, cells were collected and lysed in ice‐cold Nonidet P‐40 cell lysis buffer (10 mM Tris‐HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, and 0.4% Nonidet P‐40) with protease inhibitors and incubated on ice for 5 min, then lysate was centrifugated at 2000 × g for 5 min at 4 °C. The lysate pellet was collected and washed with lysis buffer twice, the pellet was then resuspended in 50 mL glycerol buffer (10 mM Tris‐HCl pH 7.4, 0.1 mM EDTA, 5 mM MgAc2, and 25% (vol/vol) glycerol), mixed with equal volume of MNase digestion buffer (50 mM KCl, 8 mM MgCl2, 2 mM CaCl2, and 100 mM Tris‐HCl pH 7.4), and incubated at 37 °C for 5 min with 1 U MNase (Thermo Fisher Scientific) per 100 mL of total reaction volume. The reactions were quenched by adding EDTA at the final concentration of 10 mM. The reaction products were separated by electrophoresis in 1% agarose gel.
10
Yeast Nuclei Micrococcal Nuclease Digestion
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The yeast nuclei were isolated as described (Wechser et al., 1997) . For micrococcal nuclease (MNase, Thermo Fisher Scientific) digestion, nuclei were adjusted to a DNA concentration of 200 mg/mL, and digested with 50 U/mL of MNase for various lengths of time. The reactions were terminated with 0.1 volume of stop solution (0.5 M Tris pH 8.0, 250 mM EDTA, 5% SDS). Samples were treated with 0.5 mg/mL proteinase K at 50 C for 2 h, extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and precipitated with 0.1 volume of 3M sodium acetate (pH 5.2) and 2.5 volumes of cold ethanol. Precipitated DNA was resuspended in deionized water and electrophoresed on a 1% agarose-TAE gel.
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