e coli rnase h New England Biolabs Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs e coli rnase h
    Proof of principle on construct 1. ( A ): Denaturing polyacrylamide gel showing the cleavage of a tandem transcript and comparison of transcription from a single-repeat ssDNA template. Lane 1: Tandem transcription of the construct from the linearized plasmid. Lane 2: Simultaneous <t>RNase</t> H cleavage during tandem transcription leading to the main product of 20 nt. The 12* label refers to the chimeric cleavage guide consisting of 4 DNA nucleotides and 8 2′-OMe nucleotides. Lane 3: IVT from a single-repeat template encoding construct 1 showing much higher levels of longer and shorter products. For comparison, the same amount of reaction has been loaded in all lanes. ( B ): Ion-exchange HPLC chromatogram for the 1 mL IVT from A (lane 3, construct 1*) and denaturing PAGE sampling the eluted peaks. The target peak overlaps with side products and is of low intensity. ( C ): Ion-exchange HPLC chromatogram for a 1 mL reaction of cleaved tandem transcription from A (lane 2, construct 1). The main signal is pure judging from denaturing PAGE, and of significantly larger intensity than from single-repeat template.
    E Coli Rnase H, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli rnase h/product/New England Biolabs
    Average 99 stars, based on 201 article reviews
    Price from $9.99 to $1999.99
    e coli rnase h - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs dna polymerase
    Proof of principle on construct 1. ( A ): Denaturing polyacrylamide gel showing the cleavage of a tandem transcript and comparison of transcription from a single-repeat ssDNA template. Lane 1: Tandem transcription of the construct from the linearized plasmid. Lane 2: Simultaneous <t>RNase</t> H cleavage during tandem transcription leading to the main product of 20 nt. The 12* label refers to the chimeric cleavage guide consisting of 4 DNA nucleotides and 8 2′-OMe nucleotides. Lane 3: IVT from a single-repeat template encoding construct 1 showing much higher levels of longer and shorter products. For comparison, the same amount of reaction has been loaded in all lanes. ( B ): Ion-exchange HPLC chromatogram for the 1 mL IVT from A (lane 3, construct 1*) and denaturing PAGE sampling the eluted peaks. The target peak overlaps with side products and is of low intensity. ( C ): Ion-exchange HPLC chromatogram for a 1 mL reaction of cleaved tandem transcription from A (lane 2, construct 1). The main signal is pure judging from denaturing PAGE, and of significantly larger intensity than from single-repeat template.
    Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4972 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna polymerase/product/New England Biolabs
    Average 99 stars, based on 4972 article reviews
    Price from $9.99 to $1999.99
    dna polymerase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs exonuclease 1
    Map of the MKPV genome. (A-B) Maps of the MKPV/MuCPV strains from Centenary Institute (CI, accession MH670587), Memorial Sloan Kettering Cancer Center (MSKCC, accession MH670588) and New York City basements (wild-NY, MF175078). “Bowties” indicate terminal repeats (TR). (A) Single nucleotide variations (SNV) between the CI, MSKCC and wild-NY accessions. Vertical lines—differences between accessions. Half height vertical lines—polymorphisms within an accession. ▼; 2 bp insertion in the CI strain. ▲; 1 bp insertion in a CI sub-strain. Dashed lines—missing extremities in MSKCC and wild-NY accessions, which consist of the exterior inverted repeats in the full-length CI sequence. (B-C) Alternative splicing allows production of the polypeptides p10, p15, NS1, NS2, NP and VP. Black, brown or blue shading indicate the relative reading frames of ORFs. p15, p10 and NP could theoretically be produced from multiple transcripts. Orange or red indicate peptides present in LC-MS/MS datasets PXD014938 (this paper) or PXD010540 [ 9 ], respectively. Exon or intron sequences flanking splice sites are shown in black or red text, respectively. (C) Quantitation of spliced MKPV reads in RNAseq data pooled from two MKPV-infected kidneys. Columns indicate splice site usage (left y-axis); heights of arcs (right y-axis) indicate the abundance of specific splice combinations. See S4 Table for more information. (D-E) Detection of spliced transcripts by RT-dependent PCR, using primers mapped in A-B. Input templates were MKPV-infected (D) kidney DNA or (E) <t>DNAse/ExoI-treated</t> kidney RNA, converted (+RT) or mock-converted (-RT) to cDNA. RT-PCR products corresponding to transcripts 1 to 4 are indicated by white numbers. (F) Mapping of transcription start and stop sites by RACE. See S1 Fig for RACE details. Major 5’ and 3’ RACE products, indicated by black arrows and corresponding to transcripts 2 to 4 or polyadenylation signals A and B, were gel-purified and Sanger sequenced. Other RACE products mentioned in the text are indicated by white arrows.
    Exonuclease 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease 1/product/New England Biolabs
    Average 99 stars, based on 150 article reviews
    Price from $9.99 to $1999.99
    exonuclease 1 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    84
    Thermo Fisher ambion rnase h from e coli
    Map of the MKPV genome. (A-B) Maps of the MKPV/MuCPV strains from Centenary Institute (CI, accession MH670587), Memorial Sloan Kettering Cancer Center (MSKCC, accession MH670588) and New York City basements (wild-NY, MF175078). “Bowties” indicate terminal repeats (TR). (A) Single nucleotide variations (SNV) between the CI, MSKCC and wild-NY accessions. Vertical lines—differences between accessions. Half height vertical lines—polymorphisms within an accession. ▼; 2 bp insertion in the CI strain. ▲; 1 bp insertion in a CI sub-strain. Dashed lines—missing extremities in MSKCC and wild-NY accessions, which consist of the exterior inverted repeats in the full-length CI sequence. (B-C) Alternative splicing allows production of the polypeptides p10, p15, NS1, NS2, NP and VP. Black, brown or blue shading indicate the relative reading frames of ORFs. p15, p10 and NP could theoretically be produced from multiple transcripts. Orange or red indicate peptides present in LC-MS/MS datasets PXD014938 (this paper) or PXD010540 [ 9 ], respectively. Exon or intron sequences flanking splice sites are shown in black or red text, respectively. (C) Quantitation of spliced MKPV reads in RNAseq data pooled from two MKPV-infected kidneys. Columns indicate splice site usage (left y-axis); heights of arcs (right y-axis) indicate the abundance of specific splice combinations. See S4 Table for more information. (D-E) Detection of spliced transcripts by RT-dependent PCR, using primers mapped in A-B. Input templates were MKPV-infected (D) kidney DNA or (E) <t>DNAse/ExoI-treated</t> kidney RNA, converted (+RT) or mock-converted (-RT) to cDNA. RT-PCR products corresponding to transcripts 1 to 4 are indicated by white numbers. (F) Mapping of transcription start and stop sites by RACE. See S1 Fig for RACE details. Major 5’ and 3’ RACE products, indicated by black arrows and corresponding to transcripts 2 to 4 or polyadenylation signals A and B, were gel-purified and Sanger sequenced. Other RACE products mentioned in the text are indicated by white arrows.
    Ambion Rnase H From E Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ambion rnase h from e coli/product/Thermo Fisher
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ambion rnase h from e coli - by Bioz Stars, 2020-09
    84/100 stars
      Buy from Supplier

    99
    New England Biolabs human rnase h1
    A. Schematic of the RNA:DNA hybrid transfection strategy used as a positive control for in situ S9.6 staining and <t>RNase</t> H1 enzymatic activity. B. Representative single plane image showing transfected U2OS cells containing Cy5-labeled RNA:DNA hybrids (red) that co-localize with S9.6 (green). C. Representative single plane images of mock- and RNase H1-treated transfected cells. D. Quantification of the mean S9.6 intensities of individual Cy5 foci in mock- and RNase H1-treated cells. Plots represent combined data from two biological replicates.
    Human Rnase H1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human rnase h1/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human rnase h1 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs exosap
    A. Schematic of the RNA:DNA hybrid transfection strategy used as a positive control for in situ S9.6 staining and <t>RNase</t> H1 enzymatic activity. B. Representative single plane image showing transfected U2OS cells containing Cy5-labeled RNA:DNA hybrids (red) that co-localize with S9.6 (green). C. Representative single plane images of mock- and RNase H1-treated transfected cells. D. Quantification of the mean S9.6 intensities of individual Cy5 foci in mock- and RNase H1-treated cells. Plots represent combined data from two biological replicates.
    Exosap, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exosap/product/New England Biolabs
    Average 99 stars, based on 130 article reviews
    Price from $9.99 to $1999.99
    exosap - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Proof of principle on construct 1. ( A ): Denaturing polyacrylamide gel showing the cleavage of a tandem transcript and comparison of transcription from a single-repeat ssDNA template. Lane 1: Tandem transcription of the construct from the linearized plasmid. Lane 2: Simultaneous RNase H cleavage during tandem transcription leading to the main product of 20 nt. The 12* label refers to the chimeric cleavage guide consisting of 4 DNA nucleotides and 8 2′-OMe nucleotides. Lane 3: IVT from a single-repeat template encoding construct 1 showing much higher levels of longer and shorter products. For comparison, the same amount of reaction has been loaded in all lanes. ( B ): Ion-exchange HPLC chromatogram for the 1 mL IVT from A (lane 3, construct 1*) and denaturing PAGE sampling the eluted peaks. The target peak overlaps with side products and is of low intensity. ( C ): Ion-exchange HPLC chromatogram for a 1 mL reaction of cleaved tandem transcription from A (lane 2, construct 1). The main signal is pure judging from denaturing PAGE, and of significantly larger intensity than from single-repeat template.

    Journal: Molecules

    Article Title: One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts

    doi: 10.3390/molecules25051142

    Figure Lengend Snippet: Proof of principle on construct 1. ( A ): Denaturing polyacrylamide gel showing the cleavage of a tandem transcript and comparison of transcription from a single-repeat ssDNA template. Lane 1: Tandem transcription of the construct from the linearized plasmid. Lane 2: Simultaneous RNase H cleavage during tandem transcription leading to the main product of 20 nt. The 12* label refers to the chimeric cleavage guide consisting of 4 DNA nucleotides and 8 2′-OMe nucleotides. Lane 3: IVT from a single-repeat template encoding construct 1 showing much higher levels of longer and shorter products. For comparison, the same amount of reaction has been loaded in all lanes. ( B ): Ion-exchange HPLC chromatogram for the 1 mL IVT from A (lane 3, construct 1*) and denaturing PAGE sampling the eluted peaks. The target peak overlaps with side products and is of low intensity. ( C ): Ion-exchange HPLC chromatogram for a 1 mL reaction of cleaved tandem transcription from A (lane 2, construct 1). The main signal is pure judging from denaturing PAGE, and of significantly larger intensity than from single-repeat template.

    Article Snippet: E.coli RNase H shows unimpaired enzymatic activity within buffer systems of the T7 buffer and the commercial RNase H reaction buffer (NEB).

    Techniques: Construct, Plasmid Preparation, High Performance Liquid Chromatography, Polyacrylamide Gel Electrophoresis, Sampling

    Schematic representation of the reported protocol. ( A ): (left) Tandem transcription from a linearized plasmid template with T7 RNA polymerase (T7RNAP) and (right) successive cleavage of the transcript to target length RNA by RNase H, directed by a chimeric DNA guide. ( B ): Detailed schematic of the tandem template, which starts with the viral T7RNAP promoter, an initiation sequence. The target sequence (dark blue, example here is 20 nt length) is repeated n number of times. The repeats flanked by a 5′- and 3′-spacer sequences consisting of the last eight and first four nucleotides respectively to allow for removal of the initiation and restriction sequences from the first and last repeat unit. ( C ): Hybridization of the tandem transcript (red) and the chimeric cleavage guides (green). RNase H cleaves the RNA opposite the DNA 5′-end. The 2′-OMe RNA flanks increase specificity by enhancing the binding affinity of cleavage guide to the target RNA.

    Journal: Molecules

    Article Title: One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts

    doi: 10.3390/molecules25051142

    Figure Lengend Snippet: Schematic representation of the reported protocol. ( A ): (left) Tandem transcription from a linearized plasmid template with T7 RNA polymerase (T7RNAP) and (right) successive cleavage of the transcript to target length RNA by RNase H, directed by a chimeric DNA guide. ( B ): Detailed schematic of the tandem template, which starts with the viral T7RNAP promoter, an initiation sequence. The target sequence (dark blue, example here is 20 nt length) is repeated n number of times. The repeats flanked by a 5′- and 3′-spacer sequences consisting of the last eight and first four nucleotides respectively to allow for removal of the initiation and restriction sequences from the first and last repeat unit. ( C ): Hybridization of the tandem transcript (red) and the chimeric cleavage guides (green). RNase H cleaves the RNA opposite the DNA 5′-end. The 2′-OMe RNA flanks increase specificity by enhancing the binding affinity of cleavage guide to the target RNA.

    Article Snippet: E.coli RNase H shows unimpaired enzymatic activity within buffer systems of the T7 buffer and the commercial RNase H reaction buffer (NEB).

    Techniques: Plasmid Preparation, Sequencing, Hybridization, Binding Assay

    RNase H‐deficient cells are weakly sensitive to camptothecin Plating assays indicate  rnh1 / 201∆  cells are weakly sensitive to CPT. Fivefold serial dilutions of cells were incubated on plates containing the indicated concentrations of CPT. Plates were photographed after 3‐day incubation at 32°C. Note  rad50∆  and  rnh1 / 201∆  cells form smaller colonies, indicating increased cell death. In transient exposure assays,  rnh1 / 201∆  cells are only weakly sensitive to CPT. Cells were exposed to 20 μM of CPT for 0–4 h. Bars represent standard deviation of three independent biological experiments. Plating assays indicate  rnh1 / 201∆  cells are moderately sensitive to HU. In transient exposure assays,  rnh1 / 201∆  cells are moderately sensitive to HU. Cells were treated with the indicated doses of HU for 6 h. Bars represent standard deviation of three independent biological experiments.

    Journal: EMBO Reports

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

    doi: 10.15252/embr.201745335

    Figure Lengend Snippet: RNase H‐deficient cells are weakly sensitive to camptothecin Plating assays indicate rnh1 / 201∆ cells are weakly sensitive to CPT. Fivefold serial dilutions of cells were incubated on plates containing the indicated concentrations of CPT. Plates were photographed after 3‐day incubation at 32°C. Note rad50∆ and rnh1 / 201∆ cells form smaller colonies, indicating increased cell death. In transient exposure assays, rnh1 / 201∆ cells are only weakly sensitive to CPT. Cells were exposed to 20 μM of CPT for 0–4 h. Bars represent standard deviation of three independent biological experiments. Plating assays indicate rnh1 / 201∆ cells are moderately sensitive to HU. In transient exposure assays, rnh1 / 201∆ cells are moderately sensitive to HU. Cells were treated with the indicated doses of HU for 6 h. Bars represent standard deviation of three independent biological experiments.

    Article Snippet: Beads were incubated for 2.0 h at 37°C in the absence or presence of 15 μl of recombinant Escherichia coli RNase HI (75 units, NEB, M0297L), with shaking at 1,000 rpm (Eppendorf Thermomixer).

    Techniques: Cycling Probe Technology, Incubation, Standard Deviation

    RNA–DNA hybrids are not enriched at the reparable mat1 DSB Diagram of the mat2,3∆ broken replication fork showing the location of PCR products used for ChIP and DRIP assays. Rad52 is enriched at the mat1 DSB site and the sister chromatid region used for HDR. ChIP assay was performed with Rad52‐5FLAG expressed from the endogenous locus in a mat2,3∆ strain. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments. RNA–DNA hybrids are enriched at the tRNATyr gene, but not at the reparable DSB at the mat1 locus in rnh1 / 201∆ cells. DRIP assay was performed with S9.6 antibody using the indicated strains with or without RNase H treatment. Relative enrichment was calculated as ChIP/input. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments.

    Journal: EMBO Reports

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

    doi: 10.15252/embr.201745335

    Figure Lengend Snippet: RNA–DNA hybrids are not enriched at the reparable mat1 DSB Diagram of the mat2,3∆ broken replication fork showing the location of PCR products used for ChIP and DRIP assays. Rad52 is enriched at the mat1 DSB site and the sister chromatid region used for HDR. ChIP assay was performed with Rad52‐5FLAG expressed from the endogenous locus in a mat2,3∆ strain. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments. RNA–DNA hybrids are enriched at the tRNATyr gene, but not at the reparable DSB at the mat1 locus in rnh1 / 201∆ cells. DRIP assay was performed with S9.6 antibody using the indicated strains with or without RNase H treatment. Relative enrichment was calculated as ChIP/input. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments.

    Article Snippet: Beads were incubated for 2.0 h at 37°C in the absence or presence of 15 μl of recombinant Escherichia coli RNase HI (75 units, NEB, M0297L), with shaking at 1,000 rpm (Eppendorf Thermomixer).

    Techniques: Polymerase Chain Reaction, Chromatin Immunoprecipitation

    HDR‐mediated reset of collapsed replication forks is essential in RNase H‐deficient cells Tetrad analysis showing that Rad50 is essential in  rnh1 / 201∆  background. Tetrad analysis showing that Ctp1 is essential in  rnh1 / 201∆  background. Tetrad analysis showing that Mus81 is essential in  rnh1 / 201∆  background.

    Journal: EMBO Reports

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

    doi: 10.15252/embr.201745335

    Figure Lengend Snippet: HDR‐mediated reset of collapsed replication forks is essential in RNase H‐deficient cells Tetrad analysis showing that Rad50 is essential in rnh1 / 201∆ background. Tetrad analysis showing that Ctp1 is essential in rnh1 / 201∆ background. Tetrad analysis showing that Mus81 is essential in rnh1 / 201∆ background.

    Article Snippet: Beads were incubated for 2.0 h at 37°C in the absence or presence of 15 μl of recombinant Escherichia coli RNase HI (75 units, NEB, M0297L), with shaking at 1,000 rpm (Eppendorf Thermomixer).

    Techniques:

    RNase H‐deficient cells are insensitive to ionizing radiation RNase H is not required for IR survival. Cells exposed to IR from a cesium‐137 source were plated with fivefold serial dilutions. Plates were photographed after 3‐day incubation at 32°C. Quantitative analysis confirms that rnh1 / 201∆ cells are insensitive to IR. Bars represent standard deviation of three independent biological experiments.

    Journal: EMBO Reports

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

    doi: 10.15252/embr.201745335

    Figure Lengend Snippet: RNase H‐deficient cells are insensitive to ionizing radiation RNase H is not required for IR survival. Cells exposed to IR from a cesium‐137 source were plated with fivefold serial dilutions. Plates were photographed after 3‐day incubation at 32°C. Quantitative analysis confirms that rnh1 / 201∆ cells are insensitive to IR. Bars represent standard deviation of three independent biological experiments.

    Article Snippet: Beads were incubated for 2.0 h at 37°C in the absence or presence of 15 μl of recombinant Escherichia coli RNase HI (75 units, NEB, M0297L), with shaking at 1,000 rpm (Eppendorf Thermomixer).

    Techniques: Incubation, Standard Deviation

    DSB repair at the mat1 broken replication fork occurs efficiently in the absence of RNase H Mating type switching system in fission yeast. See text for details. Lower panel shows repair mechanism in mat2,3∆ donorless strain. Proficient mating type switching in h 90 rnh1 / 201∆ cells. Colonies of the indicated genotypes on SSA plates were exposed to iodine vapor to assess mating/sporulation efficiency. Controls include wild‐type h 90 , non‐switchable h − , and switching‐defective swi3∆ h 90 . Rad50 is required SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rad50∆ mat2,3∆ cells display very poor growth compared to single mutants or wild type. Products of a tetrad dissection were photographed on successive days. RNase H is not required for SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rnh1 / 201∆ mat2,3∆ cells display no growth defect relative to rnh1∆ rnh201∆ .

    Journal: EMBO Reports

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

    doi: 10.15252/embr.201745335

    Figure Lengend Snippet: DSB repair at the mat1 broken replication fork occurs efficiently in the absence of RNase H Mating type switching system in fission yeast. See text for details. Lower panel shows repair mechanism in mat2,3∆ donorless strain. Proficient mating type switching in h 90 rnh1 / 201∆ cells. Colonies of the indicated genotypes on SSA plates were exposed to iodine vapor to assess mating/sporulation efficiency. Controls include wild‐type h 90 , non‐switchable h − , and switching‐defective swi3∆ h 90 . Rad50 is required SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rad50∆ mat2,3∆ cells display very poor growth compared to single mutants or wild type. Products of a tetrad dissection were photographed on successive days. RNase H is not required for SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rnh1 / 201∆ mat2,3∆ cells display no growth defect relative to rnh1∆ rnh201∆ .

    Article Snippet: Beads were incubated for 2.0 h at 37°C in the absence or presence of 15 μl of recombinant Escherichia coli RNase HI (75 units, NEB, M0297L), with shaking at 1,000 rpm (Eppendorf Thermomixer).

    Techniques: Dissection

    RNase H1 deletion mutant is expressed in hepatocytes from knockout mice and is catalytically inactive. ( A ) Schematic of RNase H1 mRNA with LoxP sites indicated by asterisks, and schematic of deletion mutant mRNA missing exons 4 and 5. Arrows indicate the binding sites of PCR primers. The relative positions of the three catalytic amino acids D145, E186 and D210 are indicated by a, b and c, respectively. ( B ) PCR products and ( C ) RNase H1 activity in whole liver (WL), hepatocytes (Hep) and non-parenchymal cells (NP) from RNase H1 floxed mice (Flox), constitutive knockout (cKO) mice and inducible knockout (iKO) mice. Mice were sacrificed at 6 weeks of age. iKO mice were treated with tamoxifen for 1 week prior to sacrifice. WL, Hep and NP fractions were separated from one mouse liver. The experiments were performed three times and representative results are shown.

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: RNase H1 deletion mutant is expressed in hepatocytes from knockout mice and is catalytically inactive. ( A ) Schematic of RNase H1 mRNA with LoxP sites indicated by asterisks, and schematic of deletion mutant mRNA missing exons 4 and 5. Arrows indicate the binding sites of PCR primers. The relative positions of the three catalytic amino acids D145, E186 and D210 are indicated by a, b and c, respectively. ( B ) PCR products and ( C ) RNase H1 activity in whole liver (WL), hepatocytes (Hep) and non-parenchymal cells (NP) from RNase H1 floxed mice (Flox), constitutive knockout (cKO) mice and inducible knockout (iKO) mice. Mice were sacrificed at 6 weeks of age. iKO mice were treated with tamoxifen for 1 week prior to sacrifice. WL, Hep and NP fractions were separated from one mouse liver. The experiments were performed three times and representative results are shown.

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Mutagenesis, Knock-Out, Mouse Assay, Binding Assay, Polymerase Chain Reaction, Activity Assay

    Morphology and biochemical characteristics of hepatocyte mitochondria from RNase H1 knockout mice. Liver tissue was harvested from control RNase H1 floxed mice (flox + tam) and inducible RNase H1 knockout mice (iKO + tam) 1 week post tamoxifen treatment and control RNase H1 floxed mice (flox) and constitutive RNase H1 knockout mice (cKO) at weeks 6, 14 and 26. ( A ) Transmission electron micrograph. The white arrow indicates mitochondrial fusion and the black arrow indicates mitochondrial branching. * indicates glycogen appearance. ( B ) Western blots of proteins involved in mitochondrial fusion (OPA1) and fission (DRP1). The house-keeping gene GAPDH was used as a loading control for the western analysis. Each lane shows the expression levels of these proteins in individual mice ( N = 4). Also see Supplementary Figure S1D.

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: Morphology and biochemical characteristics of hepatocyte mitochondria from RNase H1 knockout mice. Liver tissue was harvested from control RNase H1 floxed mice (flox + tam) and inducible RNase H1 knockout mice (iKO + tam) 1 week post tamoxifen treatment and control RNase H1 floxed mice (flox) and constitutive RNase H1 knockout mice (cKO) at weeks 6, 14 and 26. ( A ) Transmission electron micrograph. The white arrow indicates mitochondrial fusion and the black arrow indicates mitochondrial branching. * indicates glycogen appearance. ( B ) Western blots of proteins involved in mitochondrial fusion (OPA1) and fission (DRP1). The house-keeping gene GAPDH was used as a loading control for the western analysis. Each lane shows the expression levels of these proteins in individual mice ( N = 4). Also see Supplementary Figure S1D.

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Knock-Out, Mouse Assay, Transmission Assay, Western Blot, Expressing

    RNase H1 expression and activity in constitutive RNase H1 knockout mice as a function of age. ( A ) RNase H1 mRNA expression levels in hepatocytes from RNase H1 floxed (open circles) and cKO mice (solid circles) were determined by qRT-PCR. ( B ) Cre mRNA expression levels in hepatocytes from cKO mice (solid circles) were determined by qRT-PCR. Data are the means of four animals per group, with the exception of week 8 ( n = 2) and week 26 ( n = 3). The error bars represent ± SEM. ( C ) Gel renaturation assay showing RNase H1 activity in hepatocytes from 6, 14 and 26 week old RNase H1 floxed (Flox) and cKO mice. Each lane is representative of hepatocytes RNase H activity from one mouse. The experiments were performed three times and representative results are shown.

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: RNase H1 expression and activity in constitutive RNase H1 knockout mice as a function of age. ( A ) RNase H1 mRNA expression levels in hepatocytes from RNase H1 floxed (open circles) and cKO mice (solid circles) were determined by qRT-PCR. ( B ) Cre mRNA expression levels in hepatocytes from cKO mice (solid circles) were determined by qRT-PCR. Data are the means of four animals per group, with the exception of week 8 ( n = 2) and week 26 ( n = 3). The error bars represent ± SEM. ( C ) Gel renaturation assay showing RNase H1 activity in hepatocytes from 6, 14 and 26 week old RNase H1 floxed (Flox) and cKO mice. Each lane is representative of hepatocytes RNase H activity from one mouse. The experiments were performed three times and representative results are shown.

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Expressing, Activity Assay, Knock-Out, Mouse Assay, Quantitative RT-PCR

    RNase H1 knockout impairs liver function. Plasma levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), total bilirubin (T. Bili), triglycerides and glucose were determined for cKO mice (solid circles, solid lines, N = 4), iKO mice treated with tamoxifen beginning at week 6 (solid boxes, dashed lines, N = 4), untreated floxed mice (open circles, solid lines, N = 4) and tamoxifen-treated floxed mice (open boxes, dashed lines, N = 4). The cKO mice were monitored from ages 6 to 26 weeks of age, and the iKO mice were continuously monitored 1 week prior to tamoxifen treatment, beginning at 6 weeks of age, until death or extreme illness, which occurred at 14 weeks of age. Data are the means of four animals and ± SEM. Also see Supplementary Figure S1. GraphPad two-way ANOVA calculations were used to determine significance. * (cKO) or # (iKO) P

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: RNase H1 knockout impairs liver function. Plasma levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), total bilirubin (T. Bili), triglycerides and glucose were determined for cKO mice (solid circles, solid lines, N = 4), iKO mice treated with tamoxifen beginning at week 6 (solid boxes, dashed lines, N = 4), untreated floxed mice (open circles, solid lines, N = 4) and tamoxifen-treated floxed mice (open boxes, dashed lines, N = 4). The cKO mice were monitored from ages 6 to 26 weeks of age, and the iKO mice were continuously monitored 1 week prior to tamoxifen treatment, beginning at 6 weeks of age, until death or extreme illness, which occurred at 14 weeks of age. Data are the means of four animals and ± SEM. Also see Supplementary Figure S1. GraphPad two-way ANOVA calculations were used to determine significance. * (cKO) or # (iKO) P

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Knock-Out, AST Assay, Mouse Assay

    ASO activity impaired in RNase H1 knockout mice. ( A ) ASO activities in hepatocytes from RNase H1 floxed or knockout mice transfected with ASO at doses ranging from 40 pM to 250 nM. Hepatocytes transfected with ASO, and mRNA levels were evaluated 48-h post transfection. IC 50 correspond to the ASO concentrations resulting in 50% reduction of the target mRNA levels compared to the lipid-only treated cells. Data are the mean of triplicate determination and ± SEM. All changes between flox and knockout animals were significant, P

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: ASO activity impaired in RNase H1 knockout mice. ( A ) ASO activities in hepatocytes from RNase H1 floxed or knockout mice transfected with ASO at doses ranging from 40 pM to 250 nM. Hepatocytes transfected with ASO, and mRNA levels were evaluated 48-h post transfection. IC 50 correspond to the ASO concentrations resulting in 50% reduction of the target mRNA levels compared to the lipid-only treated cells. Data are the mean of triplicate determination and ± SEM. All changes between flox and knockout animals were significant, P

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Allele-specific Oligonucleotide, Activity Assay, Knock-Out, Mouse Assay, Transfection

    Progression of mitochondrial dysfunction and liver disease absent functional RNase H1. Summary of the observations of RNase H1 iKO and cKO mice.

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: Progression of mitochondrial dysfunction and liver disease absent functional RNase H1. Summary of the observations of RNase H1 iKO and cKO mice.

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Functional Assay, Mouse Assay

    Levels of mitochondrial DNA reduced and R-loop processing impaired in knockout livers. ( A ) Levels of mitochondrial DNA were determined by PCR. The liver tissue from the tamoxifen-treated RNase H1 floxed (hashed open bars) and iKO (hashed solid bars) mice were harvested 1 week after initiation of tamoxifen treatment. The liver tissue from RNase H1 floxed (open bars) and cKO (solid bars) mice were harvested at 6, 14 and 26 weeks of age. Data are the means of four animals per group and error bars represent ±SEM. ( B ) S7 R-Loop levels determined in hepatocytes from the tamoxifen-treated RNase H1 floxed (hashed open bars) and iKO (hashed solid bars) mice were harvested 1 week after initiation of tamoxifen treatment. Hepatocytes from RNase H1 floxed (open bars) and cKO (solid bars) mice were harvested at 6, 14 and 26 weeks of age. Data are the means of four animals per group and error bars represent ±SEM. ( C ) S7 R-Loop levels determined after treatment of the hepatocyte samples from 6-week-old RNase H1 floxed (open bars) and cKO (solid bars) mice with 90 mg (high) or 18 mg (low) of E. coli RNase H1 prior to immunoprecipitation with the heteroduplex antibody. The figure shows results from a single experiment. GraphPad two-way ANOVA calculations were used to determine significance.* P

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: Levels of mitochondrial DNA reduced and R-loop processing impaired in knockout livers. ( A ) Levels of mitochondrial DNA were determined by PCR. The liver tissue from the tamoxifen-treated RNase H1 floxed (hashed open bars) and iKO (hashed solid bars) mice were harvested 1 week after initiation of tamoxifen treatment. The liver tissue from RNase H1 floxed (open bars) and cKO (solid bars) mice were harvested at 6, 14 and 26 weeks of age. Data are the means of four animals per group and error bars represent ±SEM. ( B ) S7 R-Loop levels determined in hepatocytes from the tamoxifen-treated RNase H1 floxed (hashed open bars) and iKO (hashed solid bars) mice were harvested 1 week after initiation of tamoxifen treatment. Hepatocytes from RNase H1 floxed (open bars) and cKO (solid bars) mice were harvested at 6, 14 and 26 weeks of age. Data are the means of four animals per group and error bars represent ±SEM. ( C ) S7 R-Loop levels determined after treatment of the hepatocyte samples from 6-week-old RNase H1 floxed (open bars) and cKO (solid bars) mice with 90 mg (high) or 18 mg (low) of E. coli RNase H1 prior to immunoprecipitation with the heteroduplex antibody. The figure shows results from a single experiment. GraphPad two-way ANOVA calculations were used to determine significance.* P

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Knock-Out, Polymerase Chain Reaction, Mouse Assay, Immunoprecipitation

    ASO binding to naked 32 P-labeled SOD-1 minigene mRNA. Denaturing PAGE analysis of digestion reactions with ASOs and without ASO (labeled UTC). The bands corresponding to the RNase H1 cleavage products from on-target binding are labeled with ASO number in blue and off-target ASO cleavage products are labeled with red ASO numbers. The position of the off-target ASO hybridization in the SOD-1 minigene mRNA was determined by comparing the size of the off-target cleavage bands with the size of the on-target cleavage bands (joined by red lines).

    Journal: PLoS ONE

    Article Title: Defining the Factors That Contribute to On-Target Specificity of Antisense Oligonucleotides

    doi: 10.1371/journal.pone.0101752

    Figure Lengend Snippet: ASO binding to naked 32 P-labeled SOD-1 minigene mRNA. Denaturing PAGE analysis of digestion reactions with ASOs and without ASO (labeled UTC). The bands corresponding to the RNase H1 cleavage products from on-target binding are labeled with ASO number in blue and off-target ASO cleavage products are labeled with red ASO numbers. The position of the off-target ASO hybridization in the SOD-1 minigene mRNA was determined by comparing the size of the off-target cleavage bands with the size of the on-target cleavage bands (joined by red lines).

    Article Snippet: To each reaction, 40 U of RNaseOUT (Life Technologies, P/N#100000840) and tris(2-carboxyethyl)phosphine) (TCEP; 0.2 mM final concentration) were added, and samples were incubated for 1 h. E. coli RNase H1 cleavage reactions were initiated by adding 25 U of enzyme (New England Biolabs) to a 100 µL reaction; samples were incubated at 37°C for 60 min.

    Techniques: Allele-specific Oligonucleotide, Binding Assay, Labeling, Polyacrylamide Gel Electrophoresis, Hybridization

    Map of the MKPV genome. (A-B) Maps of the MKPV/MuCPV strains from Centenary Institute (CI, accession MH670587), Memorial Sloan Kettering Cancer Center (MSKCC, accession MH670588) and New York City basements (wild-NY, MF175078). “Bowties” indicate terminal repeats (TR). (A) Single nucleotide variations (SNV) between the CI, MSKCC and wild-NY accessions. Vertical lines—differences between accessions. Half height vertical lines—polymorphisms within an accession. ▼; 2 bp insertion in the CI strain. ▲; 1 bp insertion in a CI sub-strain. Dashed lines—missing extremities in MSKCC and wild-NY accessions, which consist of the exterior inverted repeats in the full-length CI sequence. (B-C) Alternative splicing allows production of the polypeptides p10, p15, NS1, NS2, NP and VP. Black, brown or blue shading indicate the relative reading frames of ORFs. p15, p10 and NP could theoretically be produced from multiple transcripts. Orange or red indicate peptides present in LC-MS/MS datasets PXD014938 (this paper) or PXD010540 [ 9 ], respectively. Exon or intron sequences flanking splice sites are shown in black or red text, respectively. (C) Quantitation of spliced MKPV reads in RNAseq data pooled from two MKPV-infected kidneys. Columns indicate splice site usage (left y-axis); heights of arcs (right y-axis) indicate the abundance of specific splice combinations. See S4 Table for more information. (D-E) Detection of spliced transcripts by RT-dependent PCR, using primers mapped in A-B. Input templates were MKPV-infected (D) kidney DNA or (E) DNAse/ExoI-treated kidney RNA, converted (+RT) or mock-converted (-RT) to cDNA. RT-PCR products corresponding to transcripts 1 to 4 are indicated by white numbers. (F) Mapping of transcription start and stop sites by RACE. See S1 Fig for RACE details. Major 5’ and 3’ RACE products, indicated by black arrows and corresponding to transcripts 2 to 4 or polyadenylation signals A and B, were gel-purified and Sanger sequenced. Other RACE products mentioned in the text are indicated by white arrows.

    Journal: PLoS Pathogens

    Article Title: Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys

    doi: 10.1371/journal.ppat.1008262

    Figure Lengend Snippet: Map of the MKPV genome. (A-B) Maps of the MKPV/MuCPV strains from Centenary Institute (CI, accession MH670587), Memorial Sloan Kettering Cancer Center (MSKCC, accession MH670588) and New York City basements (wild-NY, MF175078). “Bowties” indicate terminal repeats (TR). (A) Single nucleotide variations (SNV) between the CI, MSKCC and wild-NY accessions. Vertical lines—differences between accessions. Half height vertical lines—polymorphisms within an accession. ▼; 2 bp insertion in the CI strain. ▲; 1 bp insertion in a CI sub-strain. Dashed lines—missing extremities in MSKCC and wild-NY accessions, which consist of the exterior inverted repeats in the full-length CI sequence. (B-C) Alternative splicing allows production of the polypeptides p10, p15, NS1, NS2, NP and VP. Black, brown or blue shading indicate the relative reading frames of ORFs. p15, p10 and NP could theoretically be produced from multiple transcripts. Orange or red indicate peptides present in LC-MS/MS datasets PXD014938 (this paper) or PXD010540 [ 9 ], respectively. Exon or intron sequences flanking splice sites are shown in black or red text, respectively. (C) Quantitation of spliced MKPV reads in RNAseq data pooled from two MKPV-infected kidneys. Columns indicate splice site usage (left y-axis); heights of arcs (right y-axis) indicate the abundance of specific splice combinations. See S4 Table for more information. (D-E) Detection of spliced transcripts by RT-dependent PCR, using primers mapped in A-B. Input templates were MKPV-infected (D) kidney DNA or (E) DNAse/ExoI-treated kidney RNA, converted (+RT) or mock-converted (-RT) to cDNA. RT-PCR products corresponding to transcripts 1 to 4 are indicated by white numbers. (F) Mapping of transcription start and stop sites by RACE. See S1 Fig for RACE details. Major 5’ and 3’ RACE products, indicated by black arrows and corresponding to transcripts 2 to 4 or polyadenylation signals A and B, were gel-purified and Sanger sequenced. Other RACE products mentioned in the text are indicated by white arrows.

    Article Snippet: PCR, RACE and qPCR from MKPV RNA For splice site confirmations, MKPV-infected kidney RNA was treated or mock-treated with Turbo DNase (Thermo Fisher, 0.4 U/μg RNA) and ExoI (New England Biolabs, 2 U/μg RNA) for 30 min at 37˚C, followed by incubation with DNase Inactivation Reagent (Thermo Fisher, 0.2ul/ μg RNA).

    Techniques: Sequencing, Produced, Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Infection, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Purification

    DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    A. Schematic of the RNA:DNA hybrid transfection strategy used as a positive control for in situ S9.6 staining and RNase H1 enzymatic activity. B. Representative single plane image showing transfected U2OS cells containing Cy5-labeled RNA:DNA hybrids (red) that co-localize with S9.6 (green). C. Representative single plane images of mock- and RNase H1-treated transfected cells. D. Quantification of the mean S9.6 intensities of individual Cy5 foci in mock- and RNase H1-treated cells. Plots represent combined data from two biological replicates.

    Journal: bioRxiv

    Article Title: Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

    doi: 10.1101/2020.01.11.902981

    Figure Lengend Snippet: A. Schematic of the RNA:DNA hybrid transfection strategy used as a positive control for in situ S9.6 staining and RNase H1 enzymatic activity. B. Representative single plane image showing transfected U2OS cells containing Cy5-labeled RNA:DNA hybrids (red) that co-localize with S9.6 (green). C. Representative single plane images of mock- and RNase H1-treated transfected cells. D. Quantification of the mean S9.6 intensities of individual Cy5 foci in mock- and RNase H1-treated cells. Plots represent combined data from two biological replicates.

    Article Snippet: Enzymatic treatments were done in staining buffer supplemented with 3 mM magnesium chloride with 1:200 dilutions of RNase T1 (EN0541, Thermo Fisher), RNase III (ShortCut RNase III, M0245S, New England Biolabs), and/or human RNase H1 [ ] and incubated with rocking for 1 hour.

    Techniques: Transfection, Positive Control, In Situ, Staining, Activity Assay, Labeling

    A. Representative images of single planes of HeLa cells that were mock-treated or pre-treated with RNase H1, RNase III, and RNase T1 post-fixation for 1 hour at room temperature and stained with S9.6 (green) and anti-HSP27 (white). B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated.

    Journal: bioRxiv

    Article Title: Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

    doi: 10.1101/2020.01.11.902981

    Figure Lengend Snippet: A. Representative images of single planes of HeLa cells that were mock-treated or pre-treated with RNase H1, RNase III, and RNase T1 post-fixation for 1 hour at room temperature and stained with S9.6 (green) and anti-HSP27 (white). B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated.

    Article Snippet: Enzymatic treatments were done in staining buffer supplemented with 3 mM magnesium chloride with 1:200 dilutions of RNase T1 (EN0541, Thermo Fisher), RNase III (ShortCut RNase III, M0245S, New England Biolabs), and/or human RNase H1 [ ] and incubated with rocking for 1 hour.

    Techniques: Staining

    A. Genome browser tracks of a representative region of the human genome showing plus and minus strand sDRIP-seq signal obtained from mock-, RNase III-, RNase T1-, RNase T1 and III-, and RNase H1-treated DRIP samples. B. Boxplots showing the mean Pearson’s correlations of sDRIP-seq signal between mock- and enzyme-treated samples. Correlation values were calculated from data from two replicates for each condition. C. Metaplots of sDRIP-seq signal over the transcription start site (TSS), gene body, and transcription termination site (TTS) of genes with RNA expression levels in the top 10% of expressed genes for mock- and enzyme-treated samples. For the TSS and TTS, the signal was plotted over a +/− 5kb region. For gene bodies, the signal is shown as a percentile plot. Metaplots represent data from two replicates for each condition. Lines represent trimmed means and accompanying shaded areas represent standard error.

    Journal: bioRxiv

    Article Title: Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

    doi: 10.1101/2020.01.11.902981

    Figure Lengend Snippet: A. Genome browser tracks of a representative region of the human genome showing plus and minus strand sDRIP-seq signal obtained from mock-, RNase III-, RNase T1-, RNase T1 and III-, and RNase H1-treated DRIP samples. B. Boxplots showing the mean Pearson’s correlations of sDRIP-seq signal between mock- and enzyme-treated samples. Correlation values were calculated from data from two replicates for each condition. C. Metaplots of sDRIP-seq signal over the transcription start site (TSS), gene body, and transcription termination site (TTS) of genes with RNA expression levels in the top 10% of expressed genes for mock- and enzyme-treated samples. For the TSS and TTS, the signal was plotted over a +/− 5kb region. For gene bodies, the signal is shown as a percentile plot. Metaplots represent data from two replicates for each condition. Lines represent trimmed means and accompanying shaded areas represent standard error.

    Article Snippet: Enzymatic treatments were done in staining buffer supplemented with 3 mM magnesium chloride with 1:200 dilutions of RNase T1 (EN0541, Thermo Fisher), RNase III (ShortCut RNase III, M0245S, New England Biolabs), and/or human RNase H1 [ ] and incubated with rocking for 1 hour.

    Techniques: RNA Expression

    A. Ethidium bromide-stained polyacrylamide gels showing 54 nucleotide ssRNA and 54 basepair dsRNA and RNA:DNA hybrid substrates of the same sequence untreated and treated with RNase T1 and RNase III. Treatments were done for 1 hour at room temperature. B. RNA:DNA hybrids subjected to treatment with a combination of RNase T1 and III and treatment with RNase H1. C. Treatment of RNA:DNA hybrid substrates with RNase A at 0.05 mg/mL. D. Treatment of dsDNA, dsRNA, and RNA:DNA hybrids with ShortCut RNase III under manganese-supplemented conditions.

    Journal: bioRxiv

    Article Title: Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

    doi: 10.1101/2020.01.11.902981

    Figure Lengend Snippet: A. Ethidium bromide-stained polyacrylamide gels showing 54 nucleotide ssRNA and 54 basepair dsRNA and RNA:DNA hybrid substrates of the same sequence untreated and treated with RNase T1 and RNase III. Treatments were done for 1 hour at room temperature. B. RNA:DNA hybrids subjected to treatment with a combination of RNase T1 and III and treatment with RNase H1. C. Treatment of RNA:DNA hybrid substrates with RNase A at 0.05 mg/mL. D. Treatment of dsDNA, dsRNA, and RNA:DNA hybrids with ShortCut RNase III under manganese-supplemented conditions.

    Article Snippet: Enzymatic treatments were done in staining buffer supplemented with 3 mM magnesium chloride with 1:200 dilutions of RNase T1 (EN0541, Thermo Fisher), RNase III (ShortCut RNase III, M0245S, New England Biolabs), and/or human RNase H1 [ ] and incubated with rocking for 1 hour.

    Techniques: Staining, Sequencing

    Images of single planes of U2OS cells transfected with 5’-Cy5-labeled ssDNA and RNA:DNA hybrids (red) and then fixed and immunolabeled with S9.6 (green) and anti-HSP27 (white). RNA:DNA hybrid transfected cells were mock-treated and pre-treated with RNase H1 and a combination of RNase T1 and III.

    Journal: bioRxiv

    Article Title: Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

    doi: 10.1101/2020.01.11.902981

    Figure Lengend Snippet: Images of single planes of U2OS cells transfected with 5’-Cy5-labeled ssDNA and RNA:DNA hybrids (red) and then fixed and immunolabeled with S9.6 (green) and anti-HSP27 (white). RNA:DNA hybrid transfected cells were mock-treated and pre-treated with RNase H1 and a combination of RNase T1 and III.

    Article Snippet: Enzymatic treatments were done in staining buffer supplemented with 3 mM magnesium chloride with 1:200 dilutions of RNase T1 (EN0541, Thermo Fisher), RNase III (ShortCut RNase III, M0245S, New England Biolabs), and/or human RNase H1 [ ] and incubated with rocking for 1 hour.

    Techniques: Transfection, Labeling, Immunolabeling

    A. Representative images of single planes of cells that were mock-treated or pre-treated with RNase H1, RNase III, and RNase T1 for 1 hour at room temperature. B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated. Plots represent combined data from two biological replicates.

    Journal: bioRxiv

    Article Title: Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

    doi: 10.1101/2020.01.11.902981

    Figure Lengend Snippet: A. Representative images of single planes of cells that were mock-treated or pre-treated with RNase H1, RNase III, and RNase T1 for 1 hour at room temperature. B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated. Plots represent combined data from two biological replicates.

    Article Snippet: Enzymatic treatments were done in staining buffer supplemented with 3 mM magnesium chloride with 1:200 dilutions of RNase T1 (EN0541, Thermo Fisher), RNase III (ShortCut RNase III, M0245S, New England Biolabs), and/or human RNase H1 [ ] and incubated with rocking for 1 hour.

    Techniques: