rnasea  (Roche)


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    Structured Review

    Roche rnasea
    Rnasea, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnasea/product/Roche
    Average 92 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    rnasea - by Bioz Stars, 2020-09
    92/100 stars

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    Related Articles

    Protease Inhibitor:

    Article Title: HMCES Maintains Replication Fork Progression and Prevents Double-Strand Breaks in Response to APOBEC Deamination and Abasic Site Formation
    Article Snippet: .. Target cells were infected once, harvested 16 h post-infection, and lysed in 50 mM Tris-Cl pH 7.4, 1% Igepal, 0.1% sodium deoxycholate, 10% glycerol, 150 mM NaCl, 1 mM DTT, 20 μg/ml RNaseA with an EDTA-free protease inhibitor cocktail tablet (Roche). .. To test for inhibition, 16 μl each lysate (or buffer) was incubated with 100 nM of a 42 nucleotide ssDNA oligonucleotide with a single deoxyuridine and a 5′ fluorescein tag (5′ (6-FAM)-CTA TGA TGA CTC TTC TGG TCU GGA TGG TAG TTA AGT GTT GAG 3′) at 37°C and 8 μl was removed at 15 min and 30 min. As a control, 5 units of purified UDG from E. coli (New England Biolabs) was added to one reaction.

    Blocking Assay:

    Article Title: Netrin/DCC Signaling Controls Contralateral Dendrites of Octavolateralis Efferent Neurons
    Article Snippet: .. Embryos were rehydrated to PBS, washed briefly with PBST (0.1% Tween 20 in PBS), digested with 0.1% collagenase (Sigma, St. Louis, MO) for 15 min, washed with PBST, postfixed for 10 min in 4% PFA, treated with 0.25% acetic anhydride in 0.1 m triethanolamine, pH 7.0, for 1 h at RT, hybridized with the RNA probe O/N at 65°C, washed with SSCT (SSC in 0.1% Tween 20) at 65°C, treated with 10 μg/ml RNaseA at 65°C for 30 min to digest unbound probe, and incubated for at least 2 h with 2% blocking reagent (Roche). .. Embryos were then incubated O/N with alkaline phosphatase (AP)-conjugated anti-DIG Fab fragment (1:5000; Roche) and rabbit anti-GFP polyclonal antibody (pAb) (1:400; Invitrogen), washed with PBST, and developed by incubating in AP substrate (Roche) or nitroblue-tetrazolium-chloride/5-bromo-4-chlor-indolyl-phosphate (Roche) in 10% polyvinyl alcohol.

    Article Title: The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis
    Article Snippet: .. We then incubated samples in pre-hybridization solution (50% formamide, 5× saline–sodium citrate (SSC) pH 4.5, 50 μg/ml yeast RNA, 1% SDS, 50 μg/ml heparin, and nuclease-free ddH2 O) for 1 h at 68 °C, and then incubated samples in 500 ng/mL of Firre antisense riboprobe at 68 °C for 16 h. Post hybridization, samples were washed in stringency buffers and incubated in 100 μg/mL RNaseA at 37 °C for 1 h. Next, samples were washed in 1× maleic acid buffer with 0.1% Tween-20 (MBST) and incubated in Roche Blocking Reagent (Roche, 1096176) with 10% heat-inactivated sheep serum (Sigma, S2263) for 4 h at room temperature. .. We used an anti-digoxigenin antibody (Roche, 11093274910) at 1:5000 and incubated the samples for 18 h at 4 °C.

    Article Title: Manipulating a “Cocaine Engram” in Mice
    Article Snippet: .. Twenty-four hours later, sections were treated sequentially with RNaseA (1 μg/ml in 2× SSC at 37°C for 30 min), hydrogen peroxide (1% for 30 min), blocking reagent (Roche Diagnostics) with goat serum (4%, 30 min) and incubated with an anti-DIG HRP-conjugated antibody (described above) in blocking solution for 2 h at room temperature. .. Tyramide amplification was performed for 30 min at room temperature.

    Article Title: TBR2 antagonizes retinoic acid dependent neuronal differentiation by repressing ZFP423 during corticogenesis
    Article Snippet: .. Subsequently, they were treated with 10 µg/ml RNaseA for 30 min at 37°C in NTE, then washed for 4 hrs in 0.5X SSC, 20% formamide at 65°C and for 30 min in 2X SSC, and blocked for 1 hr at room temperature in 1% blocking reagent (Roche, Switzerland) in MABT. .. A 1:5000 dilution of anti-digoxigenin-AP conjugated antibody (Roche) was pre- incubated for at least 1 hr in 1% blocking reagent in MABT at 4°C.

    Purification:

    Article Title: Vitamin D receptor and STAT3 cooperate to establish TET2-mediated tolerogenesis
    Article Snippet: .. The eluate was then treated with RNaseA for 1 h at 37°C and with Proteinase K (Roche) for 1 h at 55°C and the DNA was recovered using a Qiagen PCR purification kit. .. Sequencing reads from ChIP-seq experiments were mapped to the hg19 assembly of human reference genome using Bowtie2 Aligner v2.2.6 ( ).

    Incubation:

    Article Title: Netrin/DCC Signaling Controls Contralateral Dendrites of Octavolateralis Efferent Neurons
    Article Snippet: .. Embryos were rehydrated to PBS, washed briefly with PBST (0.1% Tween 20 in PBS), digested with 0.1% collagenase (Sigma, St. Louis, MO) for 15 min, washed with PBST, postfixed for 10 min in 4% PFA, treated with 0.25% acetic anhydride in 0.1 m triethanolamine, pH 7.0, for 1 h at RT, hybridized with the RNA probe O/N at 65°C, washed with SSCT (SSC in 0.1% Tween 20) at 65°C, treated with 10 μg/ml RNaseA at 65°C for 30 min to digest unbound probe, and incubated for at least 2 h with 2% blocking reagent (Roche). .. Embryos were then incubated O/N with alkaline phosphatase (AP)-conjugated anti-DIG Fab fragment (1:5000; Roche) and rabbit anti-GFP polyclonal antibody (pAb) (1:400; Invitrogen), washed with PBST, and developed by incubating in AP substrate (Roche) or nitroblue-tetrazolium-chloride/5-bromo-4-chlor-indolyl-phosphate (Roche) in 10% polyvinyl alcohol.

    Article Title: The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis
    Article Snippet: .. We then incubated samples in pre-hybridization solution (50% formamide, 5× saline–sodium citrate (SSC) pH 4.5, 50 μg/ml yeast RNA, 1% SDS, 50 μg/ml heparin, and nuclease-free ddH2 O) for 1 h at 68 °C, and then incubated samples in 500 ng/mL of Firre antisense riboprobe at 68 °C for 16 h. Post hybridization, samples were washed in stringency buffers and incubated in 100 μg/mL RNaseA at 37 °C for 1 h. Next, samples were washed in 1× maleic acid buffer with 0.1% Tween-20 (MBST) and incubated in Roche Blocking Reagent (Roche, 1096176) with 10% heat-inactivated sheep serum (Sigma, S2263) for 4 h at room temperature. .. We used an anti-digoxigenin antibody (Roche, 11093274910) at 1:5000 and incubated the samples for 18 h at 4 °C.

    Article Title: Manipulating a “Cocaine Engram” in Mice
    Article Snippet: .. Twenty-four hours later, sections were treated sequentially with RNaseA (1 μg/ml in 2× SSC at 37°C for 30 min), hydrogen peroxide (1% for 30 min), blocking reagent (Roche Diagnostics) with goat serum (4%, 30 min) and incubated with an anti-DIG HRP-conjugated antibody (described above) in blocking solution for 2 h at room temperature. .. Tyramide amplification was performed for 30 min at room temperature.

    Infection:

    Article Title: HMCES Maintains Replication Fork Progression and Prevents Double-Strand Breaks in Response to APOBEC Deamination and Abasic Site Formation
    Article Snippet: .. Target cells were infected once, harvested 16 h post-infection, and lysed in 50 mM Tris-Cl pH 7.4, 1% Igepal, 0.1% sodium deoxycholate, 10% glycerol, 150 mM NaCl, 1 mM DTT, 20 μg/ml RNaseA with an EDTA-free protease inhibitor cocktail tablet (Roche). .. To test for inhibition, 16 μl each lysate (or buffer) was incubated with 100 nM of a 42 nucleotide ssDNA oligonucleotide with a single deoxyuridine and a 5′ fluorescein tag (5′ (6-FAM)-CTA TGA TGA CTC TTC TGG TCU GGA TGG TAG TTA AGT GTT GAG 3′) at 37°C and 8 μl was removed at 15 min and 30 min. As a control, 5 units of purified UDG from E. coli (New England Biolabs) was added to one reaction.

    Polymerase Chain Reaction:

    Article Title: Vitamin D receptor and STAT3 cooperate to establish TET2-mediated tolerogenesis
    Article Snippet: .. The eluate was then treated with RNaseA for 1 h at 37°C and with Proteinase K (Roche) for 1 h at 55°C and the DNA was recovered using a Qiagen PCR purification kit. .. Sequencing reads from ChIP-seq experiments were mapped to the hg19 assembly of human reference genome using Bowtie2 Aligner v2.2.6 ( ).

    Hybridization:

    Article Title: The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis
    Article Snippet: .. We then incubated samples in pre-hybridization solution (50% formamide, 5× saline–sodium citrate (SSC) pH 4.5, 50 μg/ml yeast RNA, 1% SDS, 50 μg/ml heparin, and nuclease-free ddH2 O) for 1 h at 68 °C, and then incubated samples in 500 ng/mL of Firre antisense riboprobe at 68 °C for 16 h. Post hybridization, samples were washed in stringency buffers and incubated in 100 μg/mL RNaseA at 37 °C for 1 h. Next, samples were washed in 1× maleic acid buffer with 0.1% Tween-20 (MBST) and incubated in Roche Blocking Reagent (Roche, 1096176) with 10% heat-inactivated sheep serum (Sigma, S2263) for 4 h at room temperature. .. We used an anti-digoxigenin antibody (Roche, 11093274910) at 1:5000 and incubated the samples for 18 h at 4 °C.

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  • 89
    Roche rnase treated
    Mature miR-17 is present in Toxoplasma -infected HFFs; mature miR-106a is absent. ( A ) The sequential <t>RNase</t> protection/primer extension assay. Paralogous miRNAs are shown as thick black lines, and RNase-labile nucleotides are shown as asterisks. ( Top box) The [5′- 32 P]-labeled oligonucleotide probe is hybridized to its targets in high-salt buffer, which forms either perfectly base-paired miRNA:probe duplexes ( left column) or miRNA:probe duplexes that are mismatched ( right column); local areas of very short and thus unstable base-pairing are indicated by dotted lines. ( Middle box) The miRNA:probe hybrids are exposed to single-strand-specific RNases (scissors), which cleave RNase-labile nucleotides in single-stranded regions of the miRNA:probe hybrids. After the initial cleavage, the mismatched miRNA:probe hybrid ( right column) is further destabilized, which exposes more single-stranded RNase-labile nucleotides in the miRNA, which are subsequently digested. ( Bottom box) The RNases and salt are removed, and the remaining miRNA:probe hybrids are subject to primer-extension analysis; extended primers are indicated by white dots. Shown are the expected extension lengths of matched and mismatched hypothetical miRNA:probe duplexes in the presence or absence of RNase. ( B ) Sequence alignment of miR-17 family members (bracket). The miR-17/106a probe sequence is shown in base-pairing orientation. Bases in bold type are RNase A– or RNase <t>T1–labile</t> nucleotides of each miRNA that do not base pair with the miR-17/106a probe; underlined uridines form dG:U base pairs with the miR-17/106a probe. ( C ) Primer-extensions were performed on synthetic templates corresponding to specific miR-17 family members. Specific primer-extension products derived from RNase-treated probe:miRNA hybrids are present only for the synthetic miR-17 and miR-106a templates (indicated with asterisks). The RNase-treated probe:miR-17 hybrid produces a primer-extension product that migrates 1 nt shorter than the corresponding product derived from the probe:miR-106a hybrid due to the presence of an RNase A–labile single-stranded C at the 5′ terminus of the probe:miR-17 hybrid. ( D ) As in C except that the template RNA was from mock-infected HFFs or from HFFs infected with Neospora or Toxoplasma for 24 h, as indicated, in the absence (−) or presence (+) of RNase A and RNase T1. Specific primer-extension products in the presence of RNase and free probe are indicated by arrows. The (−) RNA lane corresponds to primer-extensions performed in the absence of template RNA. The position of the predicted extension product specific for miR-17 (22 nt) in the presence of RNase is indicated. Size markers at 20 and 30 nt are shown.
    Rnase Treated, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase treated/product/Roche
    Average 89 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    rnase treated - by Bioz Stars, 2020-09
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    93
    Roche his tagged rnase h2
    <t>RNase</t> H2 is recruited to DSBs preferentially in the S/G2-phase of the cell cycle. a ChIP of RNASEH2A (top) and γH2AX (bottom) at the AsiSI cut site in uncut or cut DIvA cells. The bar graph shows the fold induction in cut cells (6 h after AsiSI induction) compared to uncut. Error bars represent s.e.m. ( n ≥ 2 biological replicates). b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. Scale bar: 10 μm. c Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. At least n = 200 cells were counted from four independent experiments. Lines represent mean ± s.e.m. d Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. Scale bar: 10 μm. e Dot plot shows number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. At least n = 170 cells were counted from three independent experiments. Lines represent mean ± s.e.m. f Representative pictures of super-resolution imaging analysis of γH2AX (cyan) and RNASEH2A (magenta) colocalization in G1- or S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. g Dot plot shows the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in G1- or S-phase cells. At least n = 50 events were counted from three independent experiments. Lines represent mean ± s.e.m. ** P
    His Tagged Rnase H2, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/his tagged rnase h2/product/Roche
    Average 93 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    his tagged rnase h2 - by Bioz Stars, 2020-09
    93/100 stars
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    Image Search Results


    Mature miR-17 is present in Toxoplasma -infected HFFs; mature miR-106a is absent. ( A ) The sequential RNase protection/primer extension assay. Paralogous miRNAs are shown as thick black lines, and RNase-labile nucleotides are shown as asterisks. ( Top box) The [5′- 32 P]-labeled oligonucleotide probe is hybridized to its targets in high-salt buffer, which forms either perfectly base-paired miRNA:probe duplexes ( left column) or miRNA:probe duplexes that are mismatched ( right column); local areas of very short and thus unstable base-pairing are indicated by dotted lines. ( Middle box) The miRNA:probe hybrids are exposed to single-strand-specific RNases (scissors), which cleave RNase-labile nucleotides in single-stranded regions of the miRNA:probe hybrids. After the initial cleavage, the mismatched miRNA:probe hybrid ( right column) is further destabilized, which exposes more single-stranded RNase-labile nucleotides in the miRNA, which are subsequently digested. ( Bottom box) The RNases and salt are removed, and the remaining miRNA:probe hybrids are subject to primer-extension analysis; extended primers are indicated by white dots. Shown are the expected extension lengths of matched and mismatched hypothetical miRNA:probe duplexes in the presence or absence of RNase. ( B ) Sequence alignment of miR-17 family members (bracket). The miR-17/106a probe sequence is shown in base-pairing orientation. Bases in bold type are RNase A– or RNase T1–labile nucleotides of each miRNA that do not base pair with the miR-17/106a probe; underlined uridines form dG:U base pairs with the miR-17/106a probe. ( C ) Primer-extensions were performed on synthetic templates corresponding to specific miR-17 family members. Specific primer-extension products derived from RNase-treated probe:miRNA hybrids are present only for the synthetic miR-17 and miR-106a templates (indicated with asterisks). The RNase-treated probe:miR-17 hybrid produces a primer-extension product that migrates 1 nt shorter than the corresponding product derived from the probe:miR-106a hybrid due to the presence of an RNase A–labile single-stranded C at the 5′ terminus of the probe:miR-17 hybrid. ( D ) As in C except that the template RNA was from mock-infected HFFs or from HFFs infected with Neospora or Toxoplasma for 24 h, as indicated, in the absence (−) or presence (+) of RNase A and RNase T1. Specific primer-extension products in the presence of RNase and free probe are indicated by arrows. The (−) RNA lane corresponds to primer-extensions performed in the absence of template RNA. The position of the predicted extension product specific for miR-17 (22 nt) in the presence of RNase is indicated. Size markers at 20 and 30 nt are shown.

    Journal: RNA

    Article Title: Use of two novel approaches to discriminate between closely related host microRNAs that are manipulated by Toxoplasma gondii during infection

    doi: 10.1261/rna.2069310

    Figure Lengend Snippet: Mature miR-17 is present in Toxoplasma -infected HFFs; mature miR-106a is absent. ( A ) The sequential RNase protection/primer extension assay. Paralogous miRNAs are shown as thick black lines, and RNase-labile nucleotides are shown as asterisks. ( Top box) The [5′- 32 P]-labeled oligonucleotide probe is hybridized to its targets in high-salt buffer, which forms either perfectly base-paired miRNA:probe duplexes ( left column) or miRNA:probe duplexes that are mismatched ( right column); local areas of very short and thus unstable base-pairing are indicated by dotted lines. ( Middle box) The miRNA:probe hybrids are exposed to single-strand-specific RNases (scissors), which cleave RNase-labile nucleotides in single-stranded regions of the miRNA:probe hybrids. After the initial cleavage, the mismatched miRNA:probe hybrid ( right column) is further destabilized, which exposes more single-stranded RNase-labile nucleotides in the miRNA, which are subsequently digested. ( Bottom box) The RNases and salt are removed, and the remaining miRNA:probe hybrids are subject to primer-extension analysis; extended primers are indicated by white dots. Shown are the expected extension lengths of matched and mismatched hypothetical miRNA:probe duplexes in the presence or absence of RNase. ( B ) Sequence alignment of miR-17 family members (bracket). The miR-17/106a probe sequence is shown in base-pairing orientation. Bases in bold type are RNase A– or RNase T1–labile nucleotides of each miRNA that do not base pair with the miR-17/106a probe; underlined uridines form dG:U base pairs with the miR-17/106a probe. ( C ) Primer-extensions were performed on synthetic templates corresponding to specific miR-17 family members. Specific primer-extension products derived from RNase-treated probe:miRNA hybrids are present only for the synthetic miR-17 and miR-106a templates (indicated with asterisks). The RNase-treated probe:miR-17 hybrid produces a primer-extension product that migrates 1 nt shorter than the corresponding product derived from the probe:miR-106a hybrid due to the presence of an RNase A–labile single-stranded C at the 5′ terminus of the probe:miR-17 hybrid. ( D ) As in C except that the template RNA was from mock-infected HFFs or from HFFs infected with Neospora or Toxoplasma for 24 h, as indicated, in the absence (−) or presence (+) of RNase A and RNase T1. Specific primer-extension products in the presence of RNase and free probe are indicated by arrows. The (−) RNA lane corresponds to primer-extensions performed in the absence of template RNA. The position of the predicted extension product specific for miR-17 (22 nt) in the presence of RNase is indicated. Size markers at 20 and 30 nt are shown.

    Article Snippet: For RNase-treated samples, 3.4 U RNase T1 (Roche) and 10 μg of RNase A (Sigma) were added to each 400 μL reaction mixture; and ± RNase-treated reactions were incubated for 30 min at 30°C.

    Techniques: Infection, Primer Extension Assay, Labeling, Sequencing, Derivative Assay

    VapC homologs have RNase activity and inhibit translation but a novel toxin does not. (A) Cultures of M. smegmatis harboring empty vector (pHR100) or acetamide-inducible toxin constructs were treated with 0.2% acetamide. At the indicated times, cells were labeled with of 35 S-methionine at 37°C for 1 min and incorporation of radioactivity was measured. Incorporation at t = 0 was set as 100% translation. As controls, cells were treated with 0.5 µg/ml ciprofloxacin (cip), or 25 µg/ml hygromycin (hyg). The average of three experiments is shown and error bars represent the standard deviation. (B) Cultures of M. smegmatis were grown and induced as described above. The OD 600 of each culture was measured at the indicated times. Results are plotted as fold-increase of OD 600 at each timepoint as compared to OD 600 at t = 0. The average of three experiments is shown error bars represent the standard deviation. (C) Purified toxins were incubated with MS2 RNA for 3 h at 37°C. The RNA was then purified and electrophoresed in a 1% denaturing agarose gel. Included as controls were RNA alone (RNA), His-MBP (MBP), and E. coli MazF (MazF). (D) Purified toxins MazF and Rv0301 were incubated with their respective GST-tagged antitoxins MazE (10 µg) and Rv0300 (5 µg) and 0.8 µg MS2 RNA for 3 h at 37°C. Reactions were electrophoresed in a 2% agarose gel.

    Journal: PLoS Genetics

    Article Title: Comprehensive Functional Analysis of Mycobacterium tuberculosis Toxin-Antitoxin Systems: Implications for Pathogenesis, Stress Responses, and Evolution

    doi: 10.1371/journal.pgen.1000767

    Figure Lengend Snippet: VapC homologs have RNase activity and inhibit translation but a novel toxin does not. (A) Cultures of M. smegmatis harboring empty vector (pHR100) or acetamide-inducible toxin constructs were treated with 0.2% acetamide. At the indicated times, cells were labeled with of 35 S-methionine at 37°C for 1 min and incorporation of radioactivity was measured. Incorporation at t = 0 was set as 100% translation. As controls, cells were treated with 0.5 µg/ml ciprofloxacin (cip), or 25 µg/ml hygromycin (hyg). The average of three experiments is shown and error bars represent the standard deviation. (B) Cultures of M. smegmatis were grown and induced as described above. The OD 600 of each culture was measured at the indicated times. Results are plotted as fold-increase of OD 600 at each timepoint as compared to OD 600 at t = 0. The average of three experiments is shown error bars represent the standard deviation. (C) Purified toxins were incubated with MS2 RNA for 3 h at 37°C. The RNA was then purified and electrophoresed in a 1% denaturing agarose gel. Included as controls were RNA alone (RNA), His-MBP (MBP), and E. coli MazF (MazF). (D) Purified toxins MazF and Rv0301 were incubated with their respective GST-tagged antitoxins MazE (10 µg) and Rv0300 (5 µg) and 0.8 µg MS2 RNA for 3 h at 37°C. Reactions were electrophoresed in a 2% agarose gel.

    Article Snippet: RNase activity 1.6 µg MS2 RNA (Roche) was incubated for 3 h with 1 µg of each purified protein at 37°C in 10 mM Tris-HCl (pH 7).

    Techniques: Activity Assay, Plasmid Preparation, Construct, Labeling, Radioactivity, Standard Deviation, Purification, Incubation, Agarose Gel Electrophoresis

    RNase H2 is recruited to DSBs preferentially in the S/G2-phase of the cell cycle. a ChIP of RNASEH2A (top) and γH2AX (bottom) at the AsiSI cut site in uncut or cut DIvA cells. The bar graph shows the fold induction in cut cells (6 h after AsiSI induction) compared to uncut. Error bars represent s.e.m. ( n ≥ 2 biological replicates). b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. Scale bar: 10 μm. c Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. At least n = 200 cells were counted from four independent experiments. Lines represent mean ± s.e.m. d Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. Scale bar: 10 μm. e Dot plot shows number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. At least n = 170 cells were counted from three independent experiments. Lines represent mean ± s.e.m. f Representative pictures of super-resolution imaging analysis of γH2AX (cyan) and RNASEH2A (magenta) colocalization in G1- or S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. g Dot plot shows the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in G1- or S-phase cells. At least n = 50 events were counted from three independent experiments. Lines represent mean ± s.e.m. ** P

    Journal: Nature Communications

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    doi: 10.1038/s41467-018-07799-2

    Figure Lengend Snippet: RNase H2 is recruited to DSBs preferentially in the S/G2-phase of the cell cycle. a ChIP of RNASEH2A (top) and γH2AX (bottom) at the AsiSI cut site in uncut or cut DIvA cells. The bar graph shows the fold induction in cut cells (6 h after AsiSI induction) compared to uncut. Error bars represent s.e.m. ( n ≥ 2 biological replicates). b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. Scale bar: 10 μm. c Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. At least n = 200 cells were counted from four independent experiments. Lines represent mean ± s.e.m. d Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. Scale bar: 10 μm. e Dot plot shows number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. At least n = 170 cells were counted from three independent experiments. Lines represent mean ± s.e.m. f Representative pictures of super-resolution imaging analysis of γH2AX (cyan) and RNASEH2A (magenta) colocalization in G1- or S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. g Dot plot shows the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in G1- or S-phase cells. At least n = 50 events were counted from three independent experiments. Lines represent mean ± s.e.m. ** P

    Article Snippet: Briefly, GST-BRCA2 fragments bound to Glutathione–Sepharose beads or biotin-BRC peptides bound to streptavidin beads were incubated with His-tagged RNase H2 in binding buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 1 mM PMSF, 10 mM NaF, complete protease inhibitor cocktail (Roche)) for 20 min at RT.

    Techniques: Chromatin Immunoprecipitation, Proximity Ligation Assay, Irradiation, Imaging

    BRCA2 controls DNA:RNA hybrid levels at DSBs by interacting with RNase H2 and mediating its recruitment to DSBs. a Dot plot showing the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells knocked-down for BRCA2. Scale bar: 10 μm. Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in cells knocked-down for BRCA2. At least n = 150 cells were counted from three independent experiments. Lines represent mean ± s.e.m. c Co-immunoprecipitation of endogenous RNASEH2A from not irradiated (no ir) or irradiated 5 Gy (ir) HEK293T cell extracts, prepared in the presence of benzonase to avoid contaminant nucleic acids. Asterisks indicate specific band. This experiment was repeated three times independently with similar results. d Immunoblot of GST pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to GST-tagged BRCA2 fragments. This experiment was repeated two times independently with similar results. e Immunoblot of streptavidin pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to biotinylated BRC repeats. This experiment was repeated two times independently with similar results. f Dot plot showing the normalized number of overlaps relative to random of γH2AX and DNA:RNA hybrid signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. g DRIP-qPCR at 1.5 kb on the right from the I-PpoI cut site within DAB1 gene in S/G2-phase-sorted HeLa-FUCCI cells knocked-down for BRCA2 and transfected with the I-PpoI nuclease. The bar graph shows the average fold induction of cut samples relative to uncut from n = 3 independent experiments. Error bars represent s.e.m. * P

    Journal: Nature Communications

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    doi: 10.1038/s41467-018-07799-2

    Figure Lengend Snippet: BRCA2 controls DNA:RNA hybrid levels at DSBs by interacting with RNase H2 and mediating its recruitment to DSBs. a Dot plot showing the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells knocked-down for BRCA2. Scale bar: 10 μm. Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in cells knocked-down for BRCA2. At least n = 150 cells were counted from three independent experiments. Lines represent mean ± s.e.m. c Co-immunoprecipitation of endogenous RNASEH2A from not irradiated (no ir) or irradiated 5 Gy (ir) HEK293T cell extracts, prepared in the presence of benzonase to avoid contaminant nucleic acids. Asterisks indicate specific band. This experiment was repeated three times independently with similar results. d Immunoblot of GST pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to GST-tagged BRCA2 fragments. This experiment was repeated two times independently with similar results. e Immunoblot of streptavidin pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to biotinylated BRC repeats. This experiment was repeated two times independently with similar results. f Dot plot showing the normalized number of overlaps relative to random of γH2AX and DNA:RNA hybrid signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. g DRIP-qPCR at 1.5 kb on the right from the I-PpoI cut site within DAB1 gene in S/G2-phase-sorted HeLa-FUCCI cells knocked-down for BRCA2 and transfected with the I-PpoI nuclease. The bar graph shows the average fold induction of cut samples relative to uncut from n = 3 independent experiments. Error bars represent s.e.m. * P

    Article Snippet: Briefly, GST-BRCA2 fragments bound to Glutathione–Sepharose beads or biotin-BRC peptides bound to streptavidin beads were incubated with His-tagged RNase H2 in binding buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 1 mM PMSF, 10 mM NaF, complete protease inhibitor cocktail (Roche)) for 20 min at RT.

    Techniques: Proximity Ligation Assay, Irradiation, Immunoprecipitation, Binding Assay, Recombinant, Real-time Polymerase Chain Reaction, Transfection

    RNase H2 is recruited to DSBs preferentially in the S/G2-phase of the cell cycle. a ChIP of RNASEH2A (top) and γH2AX (bottom) at the AsiSI cut site in uncut or cut DIvA cells. The bar graph shows the fold induction in cut cells (6 h after AsiSI induction) compared to uncut. Error bars represent s.e.m. ( n ≥ 2 biological replicates). b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. Scale bar: 10 μm. c Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. At least n = 200 cells were counted from four independent experiments. Lines represent mean ± s.e.m. d Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. Scale bar: 10 μm. e Dot plot shows number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. At least n = 170 cells were counted from three independent experiments. Lines represent mean ± s.e.m. f Representative pictures of super-resolution imaging analysis of γH2AX (cyan) and RNASEH2A (magenta) colocalization in G1- or S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. g Dot plot shows the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in G1- or S-phase cells. At least n = 50 events were counted from three independent experiments. Lines represent mean ± s.e.m. ** P

    Journal: Nature Communications

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    doi: 10.1038/s41467-018-07799-2

    Figure Lengend Snippet: RNase H2 is recruited to DSBs preferentially in the S/G2-phase of the cell cycle. a ChIP of RNASEH2A (top) and γH2AX (bottom) at the AsiSI cut site in uncut or cut DIvA cells. The bar graph shows the fold induction in cut cells (6 h after AsiSI induction) compared to uncut. Error bars represent s.e.m. ( n ≥ 2 biological replicates). b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. Scale bar: 10 μm. c Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. At least n = 200 cells were counted from four independent experiments. Lines represent mean ± s.e.m. d Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. Scale bar: 10 μm. e Dot plot shows number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. At least n = 170 cells were counted from three independent experiments. Lines represent mean ± s.e.m. f Representative pictures of super-resolution imaging analysis of γH2AX (cyan) and RNASEH2A (magenta) colocalization in G1- or S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. g Dot plot shows the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in G1- or S-phase cells. At least n = 50 events were counted from three independent experiments. Lines represent mean ± s.e.m. ** P

    Article Snippet: Briefly, GST-BRCA2 fragments bound to Glutathione–Sepharose beads or biotin-BRC peptides bound to streptavidin beads were incubated with His-tagged RNase H2 in binding buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 1 mM PMSF, 10 mM NaF, complete protease inhibitor cocktail (Roche)) for 20 min at RT.

    Techniques: Chromatin Immunoprecipitation, Proximity Ligation Assay, Irradiation, Imaging

    BRCA2 controls DNA:RNA hybrid levels at DSBs by interacting with RNase H2 and mediating its recruitment to DSBs. a Dot plot showing the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells knocked-down for BRCA2. Scale bar: 10 μm. Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in cells knocked-down for BRCA2. At least n = 150 cells were counted from three independent experiments. Lines represent mean ± s.e.m. c Co-immunoprecipitation of endogenous RNASEH2A from not irradiated (no ir) or irradiated 5 Gy (ir) HEK293T cell extracts, prepared in the presence of benzonase to avoid contaminant nucleic acids. Asterisks indicate specific band. This experiment was repeated three times independently with similar results. d Immunoblot of GST pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to GST-tagged BRCA2 fragments. This experiment was repeated two times independently with similar results. e Immunoblot of streptavidin pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to biotinylated BRC repeats. This experiment was repeated two times independently with similar results. f Dot plot showing the normalized number of overlaps relative to random of γH2AX and DNA:RNA hybrid signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. g DRIP-qPCR at 1.5 kb on the right from the I-PpoI cut site within DAB1 gene in S/G2-phase-sorted HeLa-FUCCI cells knocked-down for BRCA2 and transfected with the I-PpoI nuclease. The bar graph shows the average fold induction of cut samples relative to uncut from n = 3 independent experiments. Error bars represent s.e.m. * P

    Journal: Nature Communications

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    doi: 10.1038/s41467-018-07799-2

    Figure Lengend Snippet: BRCA2 controls DNA:RNA hybrid levels at DSBs by interacting with RNase H2 and mediating its recruitment to DSBs. a Dot plot showing the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells knocked-down for BRCA2. Scale bar: 10 μm. Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in cells knocked-down for BRCA2. At least n = 150 cells were counted from three independent experiments. Lines represent mean ± s.e.m. c Co-immunoprecipitation of endogenous RNASEH2A from not irradiated (no ir) or irradiated 5 Gy (ir) HEK293T cell extracts, prepared in the presence of benzonase to avoid contaminant nucleic acids. Asterisks indicate specific band. This experiment was repeated three times independently with similar results. d Immunoblot of GST pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to GST-tagged BRCA2 fragments. This experiment was repeated two times independently with similar results. e Immunoblot of streptavidin pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to biotinylated BRC repeats. This experiment was repeated two times independently with similar results. f Dot plot showing the normalized number of overlaps relative to random of γH2AX and DNA:RNA hybrid signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. g DRIP-qPCR at 1.5 kb on the right from the I-PpoI cut site within DAB1 gene in S/G2-phase-sorted HeLa-FUCCI cells knocked-down for BRCA2 and transfected with the I-PpoI nuclease. The bar graph shows the average fold induction of cut samples relative to uncut from n = 3 independent experiments. Error bars represent s.e.m. * P

    Article Snippet: Briefly, GST-BRCA2 fragments bound to Glutathione–Sepharose beads or biotin-BRC peptides bound to streptavidin beads were incubated with His-tagged RNase H2 in binding buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 1 mM PMSF, 10 mM NaF, complete protease inhibitor cocktail (Roche)) for 20 min at RT.

    Techniques: Proximity Ligation Assay, Irradiation, Immunoprecipitation, Binding Assay, Recombinant, Real-time Polymerase Chain Reaction, Transfection

    RNase H2 recruitment to DSBs occurs al later time-points after DSB induction. Bar graphs showing fold induction of a γH2AX, b BRCA1, c RPA, d BRCA2, e RNase H2, and f RAD51 signals at the nongenic AsiSI site analyzed in Fig. 1d . Lines represent mean ± s.e.m. ( n ≥ 2 biological replicates). * P

    Journal: Nature Communications

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    doi: 10.1038/s41467-018-07799-2

    Figure Lengend Snippet: RNase H2 recruitment to DSBs occurs al later time-points after DSB induction. Bar graphs showing fold induction of a γH2AX, b BRCA1, c RPA, d BRCA2, e RNase H2, and f RAD51 signals at the nongenic AsiSI site analyzed in Fig. 1d . Lines represent mean ± s.e.m. ( n ≥ 2 biological replicates). * P

    Article Snippet: Briefly, GST-BRCA2 fragments bound to Glutathione–Sepharose beads or biotin-BRC peptides bound to streptavidin beads were incubated with His-tagged RNase H2 in binding buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 1 mM PMSF, 10 mM NaF, complete protease inhibitor cocktail (Roche)) for 20 min at RT.

    Techniques: Recombinase Polymerase Amplification