dnasei  (New England Biolabs)


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    Name:
    DNase I RNase free
    Description:
    DNase I RNase free 5 000 units
    Catalog Number:
    m0303l
    Price:
    282
    Size:
    5 000 units
    Category:
    Deoxyribonucleases DNase
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    Structured Review

    New England Biolabs dnasei
    DNase I RNase free
    DNase I RNase free 5 000 units
    https://www.bioz.com/result/dnasei/product/New England Biolabs
    Average 95 stars, based on 71 article reviews
    Price from $9.99 to $1999.99
    dnasei - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Enhancer Associated Long Non-coding RNA Transcription and Gene Regulation in Experimental Models of Rickettsial Infection"

    Article Title: Enhancer Associated Long Non-coding RNA Transcription and Gene Regulation in Experimental Models of Rickettsial Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.03014

    Analysis of epigenomic signatures around transcription start site (TSS) of elncRNAs and plncRNAs. (A–D) Average normalized RPKM (reads per kilobase million) values of RNA polII, p300, DNaseI hypersensitivity site, and CCCTC binding factor (CTCF) in elncRNAs and plncRNAs, respectively; (E,F) Contrast chromatin (H3K4Me1 and H3K4Me3) and epigenetic (RNA polII, p300, DNaseI hypersensitivity and CTCF binding site) landscapes (in mouse lungs) around TSS of NONMMUT013718 and NONMMUT024103 elncRNAs, respectively. *** P ≤ 0.001 and ns = non-significant.
    Figure Legend Snippet: Analysis of epigenomic signatures around transcription start site (TSS) of elncRNAs and plncRNAs. (A–D) Average normalized RPKM (reads per kilobase million) values of RNA polII, p300, DNaseI hypersensitivity site, and CCCTC binding factor (CTCF) in elncRNAs and plncRNAs, respectively; (E,F) Contrast chromatin (H3K4Me1 and H3K4Me3) and epigenetic (RNA polII, p300, DNaseI hypersensitivity and CTCF binding site) landscapes (in mouse lungs) around TSS of NONMMUT013718 and NONMMUT024103 elncRNAs, respectively. *** P ≤ 0.001 and ns = non-significant.

    Techniques Used: Binding Assay

    2) Product Images from "Holliday junction affinity of the base excision repair factor Endo III contributes to cholera toxin phage integration"

    Article Title: Holliday junction affinity of the base excision repair factor Endo III contributes to cholera toxin phage integration

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2012.219

    Endo III blocks XerC-catalysis on pseudo-HJ and displaces it from them. ( A ) Scheme of the suicide pseudo-HJ indicating the mismatch engineered to slow down re-ligation after XerC-cleavage. KMnO 4 sensitive residues in the XerC- and XerD-binding att P(+) arms are indicated by a star. ( B ) Resolution of att P(+)/ dif suicide pseudo-HJs. Legend as in Figure 4D . ( C ) DNase I protection and ( D ) KMnO 4 sensitivity assays of the att P(+)/ dif 1 pseudo-HJ substrate. The analysed strand was labelled on its 5′ end. A scheme of the analysed strand is drawn on the left of the gels. KMnO 4 sensitive residues in the XerC- and XerD-binding sites are indicated by a star.
    Figure Legend Snippet: Endo III blocks XerC-catalysis on pseudo-HJ and displaces it from them. ( A ) Scheme of the suicide pseudo-HJ indicating the mismatch engineered to slow down re-ligation after XerC-cleavage. KMnO 4 sensitive residues in the XerC- and XerD-binding att P(+) arms are indicated by a star. ( B ) Resolution of att P(+)/ dif suicide pseudo-HJs. Legend as in Figure 4D . ( C ) DNase I protection and ( D ) KMnO 4 sensitivity assays of the att P(+)/ dif 1 pseudo-HJ substrate. The analysed strand was labelled on its 5′ end. A scheme of the analysed strand is drawn on the left of the gels. KMnO 4 sensitive residues in the XerC- and XerD-binding sites are indicated by a star.

    Techniques Used: Ligation, Binding Assay

    3) Product Images from "Comparison of Blood RNA Extraction Methods Used for Gene Expression Profiling in Amyotrophic Lateral Sclerosis"

    Article Title: Comparison of Blood RNA Extraction Methods Used for Gene Expression Profiling in Amyotrophic Lateral Sclerosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087508

    Representative Bioanalyzer electropherograms after initial RNA extractions. High quality RNA (RIN > 7.0) can be extracted from all three methods. PAXGENE however requires a DNase step as traces from initial extractions show genomic DNA contamination at high molecular weights and as a “shoulder” to the 28S peak (indicated by asterisks). Good quality RNA can be detected after globin depletion with both TEM and PAXGENE. However, PAXGENE samples showed consistently lower concentration levels, as indicated by the smaller 18S and 28S peaks (FU, fluorescent units). Traces are representative and from single samples from each extraction method.
    Figure Legend Snippet: Representative Bioanalyzer electropherograms after initial RNA extractions. High quality RNA (RIN > 7.0) can be extracted from all three methods. PAXGENE however requires a DNase step as traces from initial extractions show genomic DNA contamination at high molecular weights and as a “shoulder” to the 28S peak (indicated by asterisks). Good quality RNA can be detected after globin depletion with both TEM and PAXGENE. However, PAXGENE samples showed consistently lower concentration levels, as indicated by the smaller 18S and 28S peaks (FU, fluorescent units). Traces are representative and from single samples from each extraction method.

    Techniques Used: Transmission Electron Microscopy, Concentration Assay

    Related Articles

    Centrifugation:

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: The RNA precipitates were the next day pelleted by centrifugation (16,000 rcf for 30 min at 4°C); the pellets were washed with ice-cold 70% ethanol and air dried. .. The resulting RNA pellets were resuspended in DEPC-treated water along with the RNAse free DNAse buffer and the resulting samples (29 μl) were incubated with 1 μl (2 U) of DNAse I (2000 U/ml; NE Biolabs) at 37°C for 1 h to digest all contaminating DNAs.

    Article Title: Microcystin Biosynthesis and mcyA Expression in Geographically Distinct Microcystis Strains under Different Nitrogen, Phosphorus, and Boron Regimes
    Article Snippet: RNA Extraction, cDNA Synthesis, and Real-Time RT-PCR Microcystis cultures (15 mL) were harvested on 11th day (exponential phase) by centrifugation at 6,000 ×g for 10 min, and the cell pellets were resuspended in 1 mL TRI Reagent® (Sigma-Aldrich, USA). .. For construction of cDNA, total RNA was digested using DNase I (New England BioLabs, MA, USA) to remove genomic DNA according to the manufacturer's instructions.

    Synthesized:

    Article Title: A permissive chromatin state regulated by ZFP281-AFF3 in controlling the imprinted Meg3 polycistron
    Article Snippet: .. Total RNA was isolated with the RNeasy (Qiagen) kit, treated with DNase I (NEB) and re-purified with RNeasy. cDNAs were synthesized with High Capacity RNA-to-cDNA Kit from Applied Biosystems. .. The expression levels were measured with Fast SYBR Green Master mix from Thermo Fisher on StepOnePlus (Applied Biosystems).

    Article Title: Tomato PEPR1 ORTHOLOG RECEPTOR-LIKE KINASE1 Regulates Responses to Systemin, Necrotrophic Fungi, and Insect Herbivory
    Article Snippet: After extraction, RNA is treated with DNase I (New England Biolabs). .. The first-strand cDNA was synthesized from 2 µg of total RNA using AMV reverse transcriptase (New England Biolabs).

    Lambda DNA Preparation:

    Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori
    Article Snippet: .. TieA showed a non-specific endonuclease activity similar to DNase I as it cleaved both circular pUC19 and linearized lambda DNA, whereas Rv3131 (lane 6) as well as heat-inactivated TieA (lane 4) did not show any detectable DNA cleavage activity (Figure ). .. Furthermore, our results demonstrated a Ca2+ –Mg2+ dependent DNA cleavage activity of TieA, since EDTA and SDS markedly inhibited the cleavage activity of TieA (Figure , lanes 8, 9, 17, 18).

    Quantitative RT-PCR:

    Article Title: A permissive chromatin state regulated by ZFP281-AFF3 in controlling the imprinted Meg3 polycistron
    Article Snippet: Paragraph title: Quantitative RT-PCR and total RNA-seq library preparation ... Total RNA was isolated with the RNeasy (Qiagen) kit, treated with DNase I (NEB) and re-purified with RNeasy. cDNAs were synthesized with High Capacity RNA-to-cDNA Kit from Applied Biosystems.

    Article Title: Tomato PEPR1 ORTHOLOG RECEPTOR-LIKE KINASE1 Regulates Responses to Systemin, Necrotrophic Fungi, and Insect Herbivory
    Article Snippet: After extraction, RNA is treated with DNase I (New England Biolabs). .. RT-qPCR analysis was performed on Mx3000P real-time PCR detection system (Stratagene) using iTaq Universal SYBR Green Supermix (Bio-Rad).

    Article Title: Microcystin Biosynthesis and mcyA Expression in Geographically Distinct Microcystis Strains under Different Nitrogen, Phosphorus, and Boron Regimes
    Article Snippet: Paragraph title: 2.2. RNA Extraction, cDNA Synthesis, and Real-Time RT-PCR ... For construction of cDNA, total RNA was digested using DNase I (New England BioLabs, MA, USA) to remove genomic DNA according to the manufacturer's instructions.

    SYBR Green Assay:

    Article Title: A permissive chromatin state regulated by ZFP281-AFF3 in controlling the imprinted Meg3 polycistron
    Article Snippet: Total RNA was isolated with the RNeasy (Qiagen) kit, treated with DNase I (NEB) and re-purified with RNeasy. cDNAs were synthesized with High Capacity RNA-to-cDNA Kit from Applied Biosystems. .. The expression levels were measured with Fast SYBR Green Master mix from Thermo Fisher on StepOnePlus (Applied Biosystems).

    Article Title: Tomato PEPR1 ORTHOLOG RECEPTOR-LIKE KINASE1 Regulates Responses to Systemin, Necrotrophic Fungi, and Insect Herbivory
    Article Snippet: After extraction, RNA is treated with DNase I (New England Biolabs). .. RT-qPCR analysis was performed on Mx3000P real-time PCR detection system (Stratagene) using iTaq Universal SYBR Green Supermix (Bio-Rad).

    Incubation:

    Article Title: Differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development
    Article Snippet: .. Selected regions were washed three times with PBS and incubated in ~200 μl Accutase with DNAse I at 37°C for ~45 min. Additional mechanical dissociation was performed by triturating the tissue. ..

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death
    Article Snippet: .. For DNase I treatment, isolated NETs were incubated with DNase I (NE Biolabs) at 10 U ml−1 for 1 h before addition to SMCs. .. Live imaging of cell death was performed on SMCs stained with Calcein AM (Thermo Fisher, 1:1,000).

    Article Title: Molecular Characterization of N-glycan Degradation and Transport in Streptococcus pneumoniae and Its Contribution to Virulence
    Article Snippet: .. Nucleic acid was incubated with 5 μL DNase I buffer, 3 μL DNase I (New England Biolabs) and 1 μL Super RNaseIN (Promega) at 37°C for 1 hr. .. Cleanup of RNA was performed with a Qiagen RNeasy mini kit as per manufacturer’s instructions.

    Article Title: LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation
    Article Snippet: In vitro FTO demethylation assay The methylated nucleic acids (dsDNA, ssDNA and ssRNA) were incubated with purified FTO in 50 μl reaction buffer (10 mM Tris-HCl, 200 mM NaCl, 100 μM Fe2+ , 200 μM α-KG, 1 mM Vc) at 37°C for 0 min, 15 min, 30 min, 1 hr, 2hrs and 4hrs. .. And then the mixture was treated with 72°C for 15 min to inactivate the proteinase K. The reacted nucleic acid substrates (1 μg) were enzymatically digested into single nucleosides by mixture with 1 U DNase I or RNase A (NEB), 1 U nuclease P1 (Sigma) and 1 U alkaline phosphatase (Takara).

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: .. The resulting RNA pellets were resuspended in DEPC-treated water along with the RNAse free DNAse buffer and the resulting samples (29 μl) were incubated with 1 μl (2 U) of DNAse I (2000 U/ml; NE Biolabs) at 37°C for 1 h to digest all contaminating DNAs. .. Subsequently, 1 μg of total RNA and 8 μl of captured RNA were used for the cDNA synthesis using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Article Title: Antibiotics Stimulate Formation of Vesicles in Staphylococcus aureus in both Phage-Dependent and -Independent Fashions and via Different Routes
    Article Snippet: .. Briefly, CMVs were resuspended in phosphate-buffered saline (PBS) and were incubated for 1 h in the presence of DNase I (NEB) to degrade extravesicular DNA. .. The DNase was then inactivated at 75C° for 15 min. DNA was released from CMVs by lysis, using 0.125% Triton X-100.

    Proliferation Assay:

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death
    Article Snippet: In some experiments, SMC viability was measured using Vybrant MTT cell proliferation assay (Thermo Fisher) according to the manufacturer’s instructions and measured with a plate reader (Tecan, InfiniteF200Pro). .. For DNase I treatment, isolated NETs were incubated with DNase I (NE Biolabs) at 10 U ml−1 for 1 h before addition to SMCs.

    Expressing:

    Article Title: A permissive chromatin state regulated by ZFP281-AFF3 in controlling the imprinted Meg3 polycistron
    Article Snippet: Total RNA was isolated with the RNeasy (Qiagen) kit, treated with DNase I (NEB) and re-purified with RNeasy. cDNAs were synthesized with High Capacity RNA-to-cDNA Kit from Applied Biosystems. .. The expression levels were measured with Fast SYBR Green Master mix from Thermo Fisher on StepOnePlus (Applied Biosystems).

    Article Title: Tomato PEPR1 ORTHOLOG RECEPTOR-LIKE KINASE1 Regulates Responses to Systemin, Necrotrophic Fungi, and Insect Herbivory
    Article Snippet: Paragraph title: RNA Extraction and Analysis of Gene Expression ... After extraction, RNA is treated with DNase I (New England Biolabs).

    Derivative Assay:

    Article Title: Antibiotics Stimulate Formation of Vesicles in Staphylococcus aureus in both Phage-Dependent and -Independent Fashions and via Different Routes
    Article Snippet: The DNA loads of CMVs derived from strains NRS135 and NRS77phage stimulated with either MMC at 100 ng/ml or antibiotics at 10× MIC were quantified as described previously , with some minor modifications. .. Briefly, CMVs were resuspended in phosphate-buffered saline (PBS) and were incubated for 1 h in the presence of DNase I (NEB) to degrade extravesicular DNA.

    Infection:

    Article Title: A permissive chromatin state regulated by ZFP281-AFF3 in controlling the imprinted Meg3 polycistron
    Article Snippet: Quantitative RT-PCR and total RNA-seq library preparation Mouse ES cells V6.5 were infected with lentivirus carrying either Non-targeting shRNA or ZFP281 shRNA in the presence of 8 ug/ml of polybrene (Sigma). .. Total RNA was isolated with the RNeasy (Qiagen) kit, treated with DNase I (NEB) and re-purified with RNeasy. cDNAs were synthesized with High Capacity RNA-to-cDNA Kit from Applied Biosystems.

    Generated:

    Article Title: Molecular Characterization of N-glycan Degradation and Transport in Streptococcus pneumoniae and Its Contribution to Virulence
    Article Snippet: Nucleic acid was incubated with 5 μL DNase I buffer, 3 μL DNase I (New England Biolabs) and 1 μL Super RNaseIN (Promega) at 37°C for 1 hr. .. DNase- and RNase-free reagents were used throughout. cDNA was generated with SmartScribe reverse transcriptase according to the manufacturer’s recommendations (New England Biolabs).

    other:

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method
    Article Snippet: RNase H (Thermo Scientific) was added for RNA digestion in DNA/RNA duplexes for 1 h. After 3 repeats, the oligonucleotides were removed by DNase I (NEB).

    Article Title: Acetylation and deacetylation of Cdc25A constitutes a novel mechanism for modulating Cdc25A functions with implications for cancer
    Article Snippet: The proteins were collected on protein A/G beads, washed with RIPA buffer supplemented with protease cocktail inhibitors, 1 mM PMSF and10 units/ml of DNase I.

    Imaging:

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death
    Article Snippet: For DNase I treatment, isolated NETs were incubated with DNase I (NE Biolabs) at 10 U ml−1 for 1 h before addition to SMCs. .. Live imaging of cell death was performed on SMCs stained with Calcein AM (Thermo Fisher, 1:1,000).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: HLA-E expression and its clinical relevance in human renal cell carcinoma
    Article Snippet: RNA extraction, cDNA synthesis and qPCR Total RNA was extracted from cell pellets employing TRIzol Reagent (Life Technologies), digested with DNase I (NEB, Ipswich, MA, USA) and applied for cDNA synthesis utilizing the RevertAidTM H Minus First Strand cDNA synthesis kit (Life Technologies) as recently reported in Jasinski-Bergner and co-authors [ ]. .. The qPCR reactions were performed in a Rotorgene cycler (Qiagen, Hilden, Germany) at 95°C for 5 min followed by 40 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 40 s. HLA-E, HLA-F and HLA-G mRNA detection from biopsies were further characterized by semi-quantitative PCR [ ] using the Titan One Tube RT-PCR System (Roche Diagnostics, Heidelberg, Germany) and specific primers for HLA-G: G.257 (forward: 5′-GGAAGAGGAGACACGGAACA-3′) and G.1225 (reverse: 5′-TGAGACAGAGACGGAGACAT-3′) by Real et al., 1999 [ ], Tm 61°C and resulting PCR products were size fractionated on 1% ethidium bromide containing agarose gels [ ].

    Article Title: Molecular Characterization of N-glycan Degradation and Transport in Streptococcus pneumoniae and Its Contribution to Virulence
    Article Snippet: Paragraph title: RNA preparation and reverse transcriptase RT-PCR ... Nucleic acid was incubated with 5 μL DNase I buffer, 3 μL DNase I (New England Biolabs) and 1 μL Super RNaseIN (Promega) at 37°C for 1 hr.

    Article Title: A CD44v+ subpopulation of breast cancer stem-like cells with enhanced lung metastasis capacity
    Article Snippet: Paragraph title: Quantitative and semi-quantitative RT-PCR analyses ... For CD44 exon-specific qPCR, mRNA was subjected to genomic DNA depletion with DNase I (NEB, Ipswich, MA, USA) prior to reverse transcription.

    MTT Assay:

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death
    Article Snippet: In some experiments, SMC viability was measured using Vybrant MTT cell proliferation assay (Thermo Fisher) according to the manufacturer’s instructions and measured with a plate reader (Tecan, InfiniteF200Pro). .. For DNase I treatment, isolated NETs were incubated with DNase I (NE Biolabs) at 10 U ml−1 for 1 h before addition to SMCs.

    In Vivo:

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: Paragraph title: Yeast in vivo RNA–protein Ni2+ -pull down (RaP-NiP) assay ... The resulting RNA pellets were resuspended in DEPC-treated water along with the RNAse free DNAse buffer and the resulting samples (29 μl) were incubated with 1 μl (2 U) of DNAse I (2000 U/ml; NE Biolabs) at 37°C for 1 h to digest all contaminating DNAs.

    RNA Sequencing Assay:

    Article Title: A permissive chromatin state regulated by ZFP281-AFF3 in controlling the imprinted Meg3 polycistron
    Article Snippet: Paragraph title: Quantitative RT-PCR and total RNA-seq library preparation ... Total RNA was isolated with the RNeasy (Qiagen) kit, treated with DNase I (NEB) and re-purified with RNeasy. cDNAs were synthesized with High Capacity RNA-to-cDNA Kit from Applied Biosystems.

    Fluorescence:

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death
    Article Snippet: PI+ cells were visualized using a climate chamber fluorescence microscope (Leica, DMi8) and quantified by ImageJ software. .. For DNase I treatment, isolated NETs were incubated with DNase I (NE Biolabs) at 10 U ml−1 for 1 h before addition to SMCs.

    Methylation:

    Article Title: LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation
    Article Snippet: In vitro FTO demethylation assay The methylated nucleic acids (dsDNA, ssDNA and ssRNA) were incubated with purified FTO in 50 μl reaction buffer (10 mM Tris-HCl, 200 mM NaCl, 100 μM Fe2+ , 200 μM α-KG, 1 mM Vc) at 37°C for 0 min, 15 min, 30 min, 1 hr, 2hrs and 4hrs. .. And then the mixture was treated with 72°C for 15 min to inactivate the proteinase K. The reacted nucleic acid substrates (1 μg) were enzymatically digested into single nucleosides by mixture with 1 U DNase I or RNase A (NEB), 1 U nuclease P1 (Sigma) and 1 U alkaline phosphatase (Takara).

    Isolation:

    Article Title: Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain
    Article Snippet: Paragraph title: RNA isolation ... RNA was treated with 2U of DNase I (New England Biolabs, Ipswich, MA, USA) for 15 min at 37 °C and purified using RNA Clean & Concentrator 25 Kit (Zymo Research, Irvine, CA, USA).

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death
    Article Snippet: .. For DNase I treatment, isolated NETs were incubated with DNase I (NE Biolabs) at 10 U ml−1 for 1 h before addition to SMCs. .. Live imaging of cell death was performed on SMCs stained with Calcein AM (Thermo Fisher, 1:1,000).

    Article Title: A permissive chromatin state regulated by ZFP281-AFF3 in controlling the imprinted Meg3 polycistron
    Article Snippet: .. Total RNA was isolated with the RNeasy (Qiagen) kit, treated with DNase I (NEB) and re-purified with RNeasy. cDNAs were synthesized with High Capacity RNA-to-cDNA Kit from Applied Biosystems. .. The expression levels were measured with Fast SYBR Green Master mix from Thermo Fisher on StepOnePlus (Applied Biosystems).

    Article Title: Tomato PEPR1 ORTHOLOG RECEPTOR-LIKE KINASE1 Regulates Responses to Systemin, Necrotrophic Fungi, and Insect Herbivory
    Article Snippet: Total RNA was isolated with Trizol reagent from tomato or Arabidopsis leaf tissues according to the manufacturer’s instructions (Invitrogen). .. After extraction, RNA is treated with DNase I (New England Biolabs).

    Article Title: Microcystin Biosynthesis and mcyA Expression in Geographically Distinct Microcystis Strains under Different Nitrogen, Phosphorus, and Boron Regimes
    Article Snippet: Zirconia beads (0.5 g, 0.2 mm; Biospec, Bartlesville, OK, USA) were added to the cell suspension and cells were disrupted by vortex for 60 s. Total RNA was isolated according to the manufacturer's instructions for the reagent and resuspended in 30 μ L of DEPC-H2 O. RNA integrity was verified by agarose electrophoresis with ethidium bromide staining. .. For construction of cDNA, total RNA was digested using DNase I (New England BioLabs, MA, USA) to remove genomic DNA according to the manufacturer's instructions.

    Microscopy:

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death
    Article Snippet: PI+ cells were visualized using a climate chamber fluorescence microscope (Leica, DMi8) and quantified by ImageJ software. .. For DNase I treatment, isolated NETs were incubated with DNase I (NE Biolabs) at 10 U ml−1 for 1 h before addition to SMCs.

    Purification:

    Article Title: Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain
    Article Snippet: .. RNA was treated with 2U of DNase I (New England Biolabs, Ipswich, MA, USA) for 15 min at 37 °C and purified using RNA Clean & Concentrator 25 Kit (Zymo Research, Irvine, CA, USA). .. Isolated RNA was then subjected to two rounds of poly(A) RNA enrichment using fresh Dynabeads (Ambion) for each round.

    Article Title: LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation
    Article Snippet: Purified FTO protein were used at 0, 0.01, 0.1, 0.5, 1, 2, 4 μg as indicated. .. And then the mixture was treated with 72°C for 15 min to inactivate the proteinase K. The reacted nucleic acid substrates (1 μg) were enzymatically digested into single nucleosides by mixture with 1 U DNase I or RNase A (NEB), 1 U nuclease P1 (Sigma) and 1 U alkaline phosphatase (Takara).

    Polymerase Chain Reaction:

    Article Title: HLA-E expression and its clinical relevance in human renal cell carcinoma
    Article Snippet: RNA extraction, cDNA synthesis and qPCR Total RNA was extracted from cell pellets employing TRIzol Reagent (Life Technologies), digested with DNase I (NEB, Ipswich, MA, USA) and applied for cDNA synthesis utilizing the RevertAidTM H Minus First Strand cDNA synthesis kit (Life Technologies) as recently reported in Jasinski-Bergner and co-authors [ ]. .. The qPCR reactions were performed in a Rotorgene cycler (Qiagen, Hilden, Germany) at 95°C for 5 min followed by 40 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 40 s. HLA-E, HLA-F and HLA-G mRNA detection from biopsies were further characterized by semi-quantitative PCR [ ] using the Titan One Tube RT-PCR System (Roche Diagnostics, Heidelberg, Germany) and specific primers for HLA-G: G.257 (forward: 5′-GGAAGAGGAGACACGGAACA-3′) and G.1225 (reverse: 5′-TGAGACAGAGACGGAGACAT-3′) by Real et al., 1999 [ ], Tm 61°C and resulting PCR products were size fractionated on 1% ethidium bromide containing agarose gels [ ].

    Article Title: A CD44v+ subpopulation of breast cancer stem-like cells with enhanced lung metastasis capacity
    Article Snippet: Semi-quantitative PCR was performed with the use of TaKaRa LA Taq (Takara), and PCR products were fractionated by agarose gel electrophoresis and stained with Goldview DNA dye. .. For CD44 exon-specific qPCR, mRNA was subjected to genomic DNA depletion with DNase I (NEB, Ipswich, MA, USA) prior to reverse transcription.

    Staining:

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death
    Article Snippet: For DNase I treatment, isolated NETs were incubated with DNase I (NE Biolabs) at 10 U ml−1 for 1 h before addition to SMCs. .. Live imaging of cell death was performed on SMCs stained with Calcein AM (Thermo Fisher, 1:1,000).

    Article Title: A CD44v+ subpopulation of breast cancer stem-like cells with enhanced lung metastasis capacity
    Article Snippet: Semi-quantitative PCR was performed with the use of TaKaRa LA Taq (Takara), and PCR products were fractionated by agarose gel electrophoresis and stained with Goldview DNA dye. .. For CD44 exon-specific qPCR, mRNA was subjected to genomic DNA depletion with DNase I (NEB, Ipswich, MA, USA) prior to reverse transcription.

    Article Title: Microcystin Biosynthesis and mcyA Expression in Geographically Distinct Microcystis Strains under Different Nitrogen, Phosphorus, and Boron Regimes
    Article Snippet: Zirconia beads (0.5 g, 0.2 mm; Biospec, Bartlesville, OK, USA) were added to the cell suspension and cells were disrupted by vortex for 60 s. Total RNA was isolated according to the manufacturer's instructions for the reagent and resuspended in 30 μ L of DEPC-H2 O. RNA integrity was verified by agarose electrophoresis with ethidium bromide staining. .. For construction of cDNA, total RNA was digested using DNase I (New England BioLabs, MA, USA) to remove genomic DNA according to the manufacturer's instructions.

    Demethylation Assay:

    Article Title: LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation
    Article Snippet: Paragraph title: In vitro FTO demethylation assay ... And then the mixture was treated with 72°C for 15 min to inactivate the proteinase K. The reacted nucleic acid substrates (1 μg) were enzymatically digested into single nucleosides by mixture with 1 U DNase I or RNase A (NEB), 1 U nuclease P1 (Sigma) and 1 U alkaline phosphatase (Takara).

    Software:

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death
    Article Snippet: PI+ cells were visualized using a climate chamber fluorescence microscope (Leica, DMi8) and quantified by ImageJ software. .. For DNase I treatment, isolated NETs were incubated with DNase I (NE Biolabs) at 10 U ml−1 for 1 h before addition to SMCs.

    Real-time Polymerase Chain Reaction:

    Article Title: HLA-E expression and its clinical relevance in human renal cell carcinoma
    Article Snippet: .. RNA extraction, cDNA synthesis and qPCR Total RNA was extracted from cell pellets employing TRIzol Reagent (Life Technologies), digested with DNase I (NEB, Ipswich, MA, USA) and applied for cDNA synthesis utilizing the RevertAidTM H Minus First Strand cDNA synthesis kit (Life Technologies) as recently reported in Jasinski-Bergner and co-authors [ ]. ..

    Article Title: Tomato PEPR1 ORTHOLOG RECEPTOR-LIKE KINASE1 Regulates Responses to Systemin, Necrotrophic Fungi, and Insect Herbivory
    Article Snippet: After extraction, RNA is treated with DNase I (New England Biolabs). .. RT-qPCR analysis was performed on Mx3000P real-time PCR detection system (Stratagene) using iTaq Universal SYBR Green Supermix (Bio-Rad).

    Article Title: A CD44v+ subpopulation of breast cancer stem-like cells with enhanced lung metastasis capacity
    Article Snippet: .. For CD44 exon-specific qPCR, mRNA was subjected to genomic DNA depletion with DNase I (NEB, Ipswich, MA, USA) prior to reverse transcription. .. Trans-well invasion assays A total of 5 × 104 serum-starved cancer cells were resuspended in serum-free medium with or without 5 μ g/ml recombinant human OPN and seeded in the inserts (BD, 353504, San Jose, CA, USA) of 8 μ m pores with 3 mg/ml matrigel (BD, 354234).

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: The resulting RNA pellets were resuspended in DEPC-treated water along with the RNAse free DNAse buffer and the resulting samples (29 μl) were incubated with 1 μl (2 U) of DNAse I (2000 U/ml; NE Biolabs) at 37°C for 1 h to digest all contaminating DNAs. .. All samples were analyzed for the amounts of co-purifying uORF RNA transcripts using qPCR as follows.

    RNA Extraction:

    Article Title: HLA-E expression and its clinical relevance in human renal cell carcinoma
    Article Snippet: .. RNA extraction, cDNA synthesis and qPCR Total RNA was extracted from cell pellets employing TRIzol Reagent (Life Technologies), digested with DNase I (NEB, Ipswich, MA, USA) and applied for cDNA synthesis utilizing the RevertAidTM H Minus First Strand cDNA synthesis kit (Life Technologies) as recently reported in Jasinski-Bergner and co-authors [ ]. ..

    Article Title: Tomato PEPR1 ORTHOLOG RECEPTOR-LIKE KINASE1 Regulates Responses to Systemin, Necrotrophic Fungi, and Insect Herbivory
    Article Snippet: Paragraph title: RNA Extraction and Analysis of Gene Expression ... After extraction, RNA is treated with DNase I (New England Biolabs).

    Article Title: Microcystin Biosynthesis and mcyA Expression in Geographically Distinct Microcystis Strains under Different Nitrogen, Phosphorus, and Boron Regimes
    Article Snippet: Paragraph title: 2.2. RNA Extraction, cDNA Synthesis, and Real-Time RT-PCR ... For construction of cDNA, total RNA was digested using DNase I (New England BioLabs, MA, USA) to remove genomic DNA according to the manufacturer's instructions.

    shRNA:

    Article Title: A permissive chromatin state regulated by ZFP281-AFF3 in controlling the imprinted Meg3 polycistron
    Article Snippet: Quantitative RT-PCR and total RNA-seq library preparation Mouse ES cells V6.5 were infected with lentivirus carrying either Non-targeting shRNA or ZFP281 shRNA in the presence of 8 ug/ml of polybrene (Sigma). .. Total RNA was isolated with the RNeasy (Qiagen) kit, treated with DNase I (NEB) and re-purified with RNeasy. cDNAs were synthesized with High Capacity RNA-to-cDNA Kit from Applied Biosystems.

    Agarose Gel Electrophoresis:

    Article Title: A CD44v+ subpopulation of breast cancer stem-like cells with enhanced lung metastasis capacity
    Article Snippet: Semi-quantitative PCR was performed with the use of TaKaRa LA Taq (Takara), and PCR products were fractionated by agarose gel electrophoresis and stained with Goldview DNA dye. .. For CD44 exon-specific qPCR, mRNA was subjected to genomic DNA depletion with DNase I (NEB, Ipswich, MA, USA) prior to reverse transcription.

    In Vitro:

    Article Title: LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation
    Article Snippet: Paragraph title: In vitro FTO demethylation assay ... And then the mixture was treated with 72°C for 15 min to inactivate the proteinase K. The reacted nucleic acid substrates (1 μg) were enzymatically digested into single nucleosides by mixture with 1 U DNase I or RNase A (NEB), 1 U nuclease P1 (Sigma) and 1 U alkaline phosphatase (Takara).

    Electrophoresis:

    Article Title: Microcystin Biosynthesis and mcyA Expression in Geographically Distinct Microcystis Strains under Different Nitrogen, Phosphorus, and Boron Regimes
    Article Snippet: Zirconia beads (0.5 g, 0.2 mm; Biospec, Bartlesville, OK, USA) were added to the cell suspension and cells were disrupted by vortex for 60 s. Total RNA was isolated according to the manufacturer's instructions for the reagent and resuspended in 30 μ L of DEPC-H2 O. RNA integrity was verified by agarose electrophoresis with ethidium bromide staining. .. For construction of cDNA, total RNA was digested using DNase I (New England BioLabs, MA, USA) to remove genomic DNA according to the manufacturer's instructions.

    Spectrophotometry:

    Article Title: Microcystin Biosynthesis and mcyA Expression in Geographically Distinct Microcystis Strains under Different Nitrogen, Phosphorus, and Boron Regimes
    Article Snippet: The quantity and quality of RNA were assessed with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc.). .. For construction of cDNA, total RNA was digested using DNase I (New England BioLabs, MA, USA) to remove genomic DNA according to the manufacturer's instructions.

    Concentration Assay:

    Article Title: Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain
    Article Snippet: RNA was treated with 2U of DNase I (New England Biolabs, Ipswich, MA, USA) for 15 min at 37 °C and purified using RNA Clean & Concentrator 25 Kit (Zymo Research, Irvine, CA, USA). .. RNA quality was assessed using an Agilent 2100 Bioanalyzer, and concentration was determined by measuring absorbance at 260 nm and 280 nm in a UV/vis-spectrophotometer.

    Article Title: Tomato PEPR1 ORTHOLOG RECEPTOR-LIKE KINASE1 Regulates Responses to Systemin, Necrotrophic Fungi, and Insect Herbivory
    Article Snippet: After extraction, RNA is treated with DNase I (New England Biolabs). .. RNA concentration was measured using Nanodrop 2000c (Thermo Scientific).

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: The Proteinase K digested Eluates as well as WCEs from the un-cross-linked cells were adjusted to 400 μl with the TES solution prepared in DEPC-treated nano-pure water to a final concentration of 10 mM Tris–Cl, pH 7.5, 10 mM EDTA, 0.5 % SDS and incubated with acid phenol in 1:1 ratio at 65°C for 10 min with vigorous vortexing for 10 s every 5 min. .. The resulting RNA pellets were resuspended in DEPC-treated water along with the RNAse free DNAse buffer and the resulting samples (29 μl) were incubated with 1 μl (2 U) of DNAse I (2000 U/ml; NE Biolabs) at 37°C for 1 h to digest all contaminating DNAs.

    Lysis:

    Article Title: Antibiotics Stimulate Formation of Vesicles in Staphylococcus aureus in both Phage-Dependent and -Independent Fashions and via Different Routes
    Article Snippet: Briefly, CMVs were resuspended in phosphate-buffered saline (PBS) and were incubated for 1 h in the presence of DNase I (NEB) to degrade extravesicular DNA. .. The DNase was then inactivated at 75C° for 15 min. DNA was released from CMVs by lysis, using 0.125% Triton X-100.

    Activity Assay:

    Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori
    Article Snippet: .. TieA showed a non-specific endonuclease activity similar to DNase I as it cleaved both circular pUC19 and linearized lambda DNA, whereas Rv3131 (lane 6) as well as heat-inactivated TieA (lane 4) did not show any detectable DNA cleavage activity (Figure ). .. Furthermore, our results demonstrated a Ca2+ –Mg2+ dependent DNA cleavage activity of TieA, since EDTA and SDS markedly inhibited the cleavage activity of TieA (Figure , lanes 8, 9, 17, 18).

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    New England Biolabs dnase i
    ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and <t>DNase</t> I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.
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    ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and DNase I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.

    Journal: Nucleic Acids Research

    Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori

    doi: 10.1093/nar/gkw730

    Figure Lengend Snippet: ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and DNase I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.

    Article Snippet: TieA showed a non-specific endonuclease activity similar to DNase I as it cleaved both circular pUC19 and linearized lambda DNA, whereas Rv3131 (lane 6) as well as heat-inactivated TieA (lane 4) did not show any detectable DNA cleavage activity (Figure ).

    Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Electrophoresis, Clear Native PAGE, Autoradiography, Activity Assay, Incubation, Agarose Gel Electrophoresis, Negative Control, Lambda DNA Preparation

    ( A ) Measurement of cell death by Annexin V-FITC/PI staining. AGS cells were infected (WT and KO strains of H. pylori ) or treated (5 μg of TieA protein) for 24 h as indicated and were examined for apoptotic cells using Annexin V-FITC apoptosis detection kit as described in materials and methods section. Staurosporine and DNase I treated AGS cells were used as positive controls. ( B ) Annexin V-FITC/PI staining to analyze apoptosis in AGS cell line induced by TieA using flow cytometry. ( C ) AGS cells were infected with H. pylori (WT or KO) at an MOI of 100 for 24 h, and cell death was measured as fold change in histone release. Data are mean ± SD of three independent experiments; two-tailed Student's t test was performed for statistical analysis, * P ≤ 0.05. ( D ) Flow cytometry analysis showing expression of Fas receptors on AGS cells upon treatment with TieA (5 μg) after 24 h. The shift in the histogram peak for TieA and staurosporine as compared to the untreated AGS cells indicates an enhanced expression of Fas receptors on AGS cells. HI: heat inactivated.

    Journal: Nucleic Acids Research

    Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori

    doi: 10.1093/nar/gkw730

    Figure Lengend Snippet: ( A ) Measurement of cell death by Annexin V-FITC/PI staining. AGS cells were infected (WT and KO strains of H. pylori ) or treated (5 μg of TieA protein) for 24 h as indicated and were examined for apoptotic cells using Annexin V-FITC apoptosis detection kit as described in materials and methods section. Staurosporine and DNase I treated AGS cells were used as positive controls. ( B ) Annexin V-FITC/PI staining to analyze apoptosis in AGS cell line induced by TieA using flow cytometry. ( C ) AGS cells were infected with H. pylori (WT or KO) at an MOI of 100 for 24 h, and cell death was measured as fold change in histone release. Data are mean ± SD of three independent experiments; two-tailed Student's t test was performed for statistical analysis, * P ≤ 0.05. ( D ) Flow cytometry analysis showing expression of Fas receptors on AGS cells upon treatment with TieA (5 μg) after 24 h. The shift in the histogram peak for TieA and staurosporine as compared to the untreated AGS cells indicates an enhanced expression of Fas receptors on AGS cells. HI: heat inactivated.

    Article Snippet: TieA showed a non-specific endonuclease activity similar to DNase I as it cleaved both circular pUC19 and linearized lambda DNA, whereas Rv3131 (lane 6) as well as heat-inactivated TieA (lane 4) did not show any detectable DNA cleavage activity (Figure ).

    Techniques: Staining, Infection, Flow Cytometry, Cytometry, Two Tailed Test, Expressing

    Schematic overview of the modified protocol.  a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified.  b , bioinformatics analysis

    Journal: BMC Genomics

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method

    doi: 10.1186/s12864-017-3725-3

    Figure Lengend Snippet: Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis

    Article Snippet: RNase H (Thermo Scientific) was added for RNA digestion in DNA/RNA duplexes for 1 h. After 3 repeats, the oligonucleotides were removed by DNase I (NEB).

    Techniques: Modification, Irradiation

    Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n = 5/group). * p

    Journal: Frontiers in Immunology

    Article Title: Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis

    doi: 10.3389/fimmu.2018.01680

    Figure Lengend Snippet: Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n = 5/group). * p

    Article Snippet: Treatment with DNase I did not modify rates of weight gain (or spleen weight/body weight ratios) (Figures S4A,B in Supplementary Material).

    Techniques: Mouse Assay, Immunofluorescence, Microscopy, Staining

    Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis

    doi: 10.3389/fimmu.2018.01680

    Figure Lengend Snippet: Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p

    Article Snippet: Treatment with DNase I did not modify rates of weight gain (or spleen weight/body weight ratios) (Figures S4A,B in Supplementary Material).

    Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Mouse Assay, Staining, Immunofluorescence, Microscopy