rnaseh  (New England Biolabs)


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    Name:
    RNase H
    Description:
    RNase H 1 250 units
    Catalog Number:
    m0297l
    Price:
    283
    Size:
    1 250 units
    Category:
    Ribonucleases RNase
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    Structured Review

    New England Biolabs rnaseh
    RNase H
    RNase H 1 250 units
    https://www.bioz.com/result/rnaseh/product/New England Biolabs
    Average 95 stars, based on 56 article reviews
    Price from $9.99 to $1999.99
    rnaseh - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Chromatin conformation regulates the coordination between DNA replication and transcription"

    Article Title: Chromatin conformation regulates the coordination between DNA replication and transcription

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03539-8

    H1-TKO cells display impaired transcription dynamics. a Diagram of the experimental design to measure transcription elongation rates by transient inhibition of initiating RNAPIIs with DRB. Three hours after DRB incubation, the drug was washed-off to resume transcription elongation and total RNA was extracted from identical number of cells at the indicated time-points (open triangles). Global nascent transcription was evaluated by 1h-EU labelling at the indicated time points (red lines). b Time course transcription elongation measurements at the Med13l and Inpp5a genes in WT mES (upper panels) and H1-TKO mES cells (lower panels). Levels of pre-mRNA at the indicated times were determined by RT-qPCR at the positions marked in the gene maps above the graphs. Pre-mRNA values were normalized to the values of the non-DRB-treated sample. Results are shown as means ± s.d. from two independent experiments ( n = 2). c Representative images of EU staining (top) and distribution of EU nuclear intensity during DRB treatment and upon drug-release at the time points shown in the experimental scheme in a . d Representative images of S9.6 immunostaining ±RNAseA or +-RNAseH incubation (top) and distribution of S9.6 nuclear intensity (bottom) in WT and H1-TKO cells. Scale bar, 10 μm. Nuclear segmentation (white lines) was based on DAPI staining. Median values are indicated ( n c-d for S-phase distribution of S9.6 and γH2AX nuclear intensities. Differences between distributions were assessed with the Mann–Whitney rank sum test. **** P
    Figure Legend Snippet: H1-TKO cells display impaired transcription dynamics. a Diagram of the experimental design to measure transcription elongation rates by transient inhibition of initiating RNAPIIs with DRB. Three hours after DRB incubation, the drug was washed-off to resume transcription elongation and total RNA was extracted from identical number of cells at the indicated time-points (open triangles). Global nascent transcription was evaluated by 1h-EU labelling at the indicated time points (red lines). b Time course transcription elongation measurements at the Med13l and Inpp5a genes in WT mES (upper panels) and H1-TKO mES cells (lower panels). Levels of pre-mRNA at the indicated times were determined by RT-qPCR at the positions marked in the gene maps above the graphs. Pre-mRNA values were normalized to the values of the non-DRB-treated sample. Results are shown as means ± s.d. from two independent experiments ( n = 2). c Representative images of EU staining (top) and distribution of EU nuclear intensity during DRB treatment and upon drug-release at the time points shown in the experimental scheme in a . d Representative images of S9.6 immunostaining ±RNAseA or +-RNAseH incubation (top) and distribution of S9.6 nuclear intensity (bottom) in WT and H1-TKO cells. Scale bar, 10 μm. Nuclear segmentation (white lines) was based on DAPI staining. Median values are indicated ( n c-d for S-phase distribution of S9.6 and γH2AX nuclear intensities. Differences between distributions were assessed with the Mann–Whitney rank sum test. **** P

    Techniques Used: Inhibition, Incubation, Quantitative RT-PCR, Staining, Immunostaining, MANN-WHITNEY

    2) Product Images from "Aging of Xenopus tropicalis Eggs Leads to Deadenylation of a Specific Set of Maternal mRNAs and Loss of Developmental Potential"

    Article Title: Aging of Xenopus tropicalis Eggs Leads to Deadenylation of a Specific Set of Maternal mRNAs and Loss of Developmental Potential

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013532

    Poly(A) tail reduction of specific maternal mRNA in aged eggs. Poly(A) tail behavior of the indicated transcripts decreased (A) and not changed (B) upon egg aging are shown. Total mRNA from fresh (0 h) and aged (3 h) eggs was assayed by the RNA ligation-mediated poly(A) test (RL-PAT). * indicates RNaseH/oligo(dT) 20 digestion prior to ligation. Control lanes: –Lig, Ligation reaction performed without RNA; -RT, ligated RNA was not reverse transcribed prior to PCR. M, DNA size marker are given in base pairs. Direct sequencing of atp5a1 and tpi1 (A lower panel) reveals the actual transcript 3′ending (indicated by arrows), which is in fresh eggs at the end of the poly(A) tail (italic As), but in aged eggs several nucleotides upstream of the former end of the RNA body (clear box). P1 is the ligated primer.
    Figure Legend Snippet: Poly(A) tail reduction of specific maternal mRNA in aged eggs. Poly(A) tail behavior of the indicated transcripts decreased (A) and not changed (B) upon egg aging are shown. Total mRNA from fresh (0 h) and aged (3 h) eggs was assayed by the RNA ligation-mediated poly(A) test (RL-PAT). * indicates RNaseH/oligo(dT) 20 digestion prior to ligation. Control lanes: –Lig, Ligation reaction performed without RNA; -RT, ligated RNA was not reverse transcribed prior to PCR. M, DNA size marker are given in base pairs. Direct sequencing of atp5a1 and tpi1 (A lower panel) reveals the actual transcript 3′ending (indicated by arrows), which is in fresh eggs at the end of the poly(A) tail (italic As), but in aged eggs several nucleotides upstream of the former end of the RNA body (clear box). P1 is the ligated primer.

    Techniques Used: Ligation, Polymerase Chain Reaction, Marker, Sequencing

    Related Articles

    Amplification:

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning
    Article Snippet: Then, DNA–RNA hybrids were digested with 5 U of RNAse H (NEB) for 15 min at 37°C, extracted with phenol and ethanol precipitated. .. Finally, the resulting cDNA was amplified by PCR using oligo(dT) and oligo 9 primers, and sequenced.

    Article Title: Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus
    Article Snippet: After 5 min, 2 μl of an enzyme mixture containing 2.6 μg of bovine serum albumin (Promega Co.), 40 international units (IU) of T7 RNA polymerase (Novagen Inc., Madison, WI), 8 IU of avian myeloblastosis virus reverse transcriptase (Seikagaku, Ijamsville, Md.), 0.2 IU of RNase H (New England Biolabs, Beverly, MA), and 12.5 IU of RNasin (Amersham Biotech) were added to each well, followed by incubation at 40° ± 1°C for 150 min. Fluorescence intensity data were recorded every minute of the NASBA reaction. .. The threshold cycle of each amplification reaction was calculated based on the first cycle at which the fluorescence was 10-fold higher than the standard deviation of the mean baseline emission.

    Article Title: Constitutive smooth muscle tumour necrosis factor regulates microvascular myogenic responsiveness and systemic blood pressure
    Article Snippet: .. Immediately following the RT reaction, RNA was removed by adding 2.5 U RNase H (New England Biolabs) and incubating the reaction at 37 °C for 20 min; the RNAse enzyme was subsequently inactivated by heating to 65 °C for 15 min. Target genes were amplified with an i-Taq DNA polymerase kit (FroggaBio). ..

    DNA Ligation:

    Article Title: The wright stuff: reimagining path analysis reveals novel components of the sex determination hierarchy in drosophila melanogaster
    Article Snippet: Second strand cDNA was synthesized using DNA polymerase I in combination with ribonuclease H (NEB). .. Illumina sequencing adapters were then ligated to both ends of the cDNA templates using Fast-Link™ DNA Ligation Kit (Epicentre).

    Synthesized:

    Article Title: Mechanical Genomic Studies Reveal the Role of d-Alanine Metabolism in Pseudomonas aeruginosa Cell Stiffness
    Article Snippet: .. Newly synthesized cDNA was treated with New England Biolabs RNase H to digest RNA hybridized to cDNA. .. Thermo Fisher PowerUp SYBR green master mix was used for quantitative PCR on an Applied Biosystems 7500 Fast real-time PCR system, following the manufacturer’s instructions for a standard cycling protocol.

    Article Title: Identification and Expression Analysis of Candidate Odorant-Binding Protein and Chemosensory Protein Genes by Antennal Transcriptome of Sitobion avenae
    Article Snippet: .. Single-stranded (ss) cDNA was synthesized using a random hexamer primer, M-MuLV Reverse Transcriptase and DNA Polymerase I and RNase H (NEB, USA). .. The 3' ends of the DNA fragments were adenylated and the NEBNext Adaptor was ligated to the fragments for hybridization.

    Article Title: The wright stuff: reimagining path analysis reveals novel components of the sex determination hierarchy in drosophila melanogaster
    Article Snippet: .. Second strand cDNA was synthesized using DNA polymerase I in combination with ribonuclease H (NEB). ..

    Quantitative RT-PCR:

    Article Title: Chlorhexidine Induces VanA-Type Vancomycin Resistance Genes in Enterococci
    Article Snippet: Paragraph title: RT-qPCR. ... RNase H (NEB) was added to remove RNA, and cDNA was purified using the QIAquick PCR purification kit (Qiagen).

    SYBR Green Assay:

    Article Title: Mechanical Genomic Studies Reveal the Role of d-Alanine Metabolism in Pseudomonas aeruginosa Cell Stiffness
    Article Snippet: Newly synthesized cDNA was treated with New England Biolabs RNase H to digest RNA hybridized to cDNA. .. Thermo Fisher PowerUp SYBR green master mix was used for quantitative PCR on an Applied Biosystems 7500 Fast real-time PCR system, following the manufacturer’s instructions for a standard cycling protocol.

    Article Title: Chlorhexidine Induces VanA-Type Vancomycin Resistance Genes in Enterococci
    Article Snippet: RNase H (NEB) was added to remove RNA, and cDNA was purified using the QIAquick PCR purification kit (Qiagen). .. RT-qPCR was performed with a Cepheid Smart Cycler and SYBR green I (Sigma-Aldrich). vanA and - B gene expression was internally normalized to clpX .

    Random Hexamer Labeling:

    Article Title: Identification and Expression Analysis of Candidate Odorant-Binding Protein and Chemosensory Protein Genes by Antennal Transcriptome of Sitobion avenae
    Article Snippet: .. Single-stranded (ss) cDNA was synthesized using a random hexamer primer, M-MuLV Reverse Transcriptase and DNA Polymerase I and RNase H (NEB, USA). .. The 3' ends of the DNA fragments were adenylated and the NEBNext Adaptor was ligated to the fragments for hybridization.

    Article Title: The wright stuff: reimagining path analysis reveals novel components of the sex determination hierarchy in drosophila melanogaster
    Article Snippet: First strand cDNA was synthesized using SuperScript® II Reverse Transcriptase (Invitrogen) and a combination of random hexamer and oligo (dT) primers. .. Second strand cDNA was synthesized using DNA polymerase I in combination with ribonuclease H (NEB).

    Standard Deviation:

    Article Title: Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus
    Article Snippet: After 5 min, 2 μl of an enzyme mixture containing 2.6 μg of bovine serum albumin (Promega Co.), 40 international units (IU) of T7 RNA polymerase (Novagen Inc., Madison, WI), 8 IU of avian myeloblastosis virus reverse transcriptase (Seikagaku, Ijamsville, Md.), 0.2 IU of RNase H (New England Biolabs, Beverly, MA), and 12.5 IU of RNasin (Amersham Biotech) were added to each well, followed by incubation at 40° ± 1°C for 150 min. Fluorescence intensity data were recorded every minute of the NASBA reaction. .. The threshold cycle of each amplification reaction was calculated based on the first cycle at which the fluorescence was 10-fold higher than the standard deviation of the mean baseline emission.

    Expressing:

    Article Title: Chlorhexidine Induces VanA-Type Vancomycin Resistance Genes in Enterococci
    Article Snippet: RNase H (NEB) was added to remove RNA, and cDNA was purified using the QIAquick PCR purification kit (Qiagen). .. RT-qPCR was performed with a Cepheid Smart Cycler and SYBR green I (Sigma-Aldrich). vanA and - B gene expression was internally normalized to clpX .

    Touchdown PCR:

    Article Title: Growth Phase and (p)ppGpp Control of IraD, a Regulator of RpoS Stability, in Escherichia coli ▿
    Article Snippet: The 5′ ends of iraD mRNA were mapped by using 5′ RACE (rapid amplification of cDNA ends) as previously described ( ) with the following modifications. cDNA was prepared by incubation of 2 μg of total RNA (isolated as described below) with 200 U of Moloney murine leukemia virus reverse transcriptase (NEB), 2 mM deoxynucleoside triphosphate, 40 μM iraD ORF specific primer (5′-TTA GCT GAC ATT CTC CAG CGT CGC ACT GCG-3′), and 1× Moloney murine leukemia virus RT buffer at 42°C for 1 h, followed by treatment with 10 μg of RNase A (Sigma)/ml and 5 U of RNase H (NEB) at 37°C for 30 min. cDNA was purified by using a QIAquick PCR purification kit (Qiagen). .. After ligation of cDNA with an anchor oligonucleotide (5′-TTT AGT GAG GGT TAA TAA GCG GCC GCG TCG TGA CTG GGA GCG C-3′), touchdown PCR was performed by using 1 U of Phusion polymerase (Finnzyme), 12 ng of purified anchored-cDNA, 0.5 μM anchor, and iraD ORF specific primers (5′-CTC CCA GTC ACG ACG CGG CCG C-3′ and 5′-CAA ACG TTA GCG GTT CAT CG-3′).

    Activated Clotting Time Assay:

    Article Title: Growth Phase and (p)ppGpp Control of IraD, a Regulator of RpoS Stability, in Escherichia coli ▿
    Article Snippet: .. The 5′ ends of iraD mRNA were mapped by using 5′ RACE (rapid amplification of cDNA ends) as previously described ( ) with the following modifications. cDNA was prepared by incubation of 2 μg of total RNA (isolated as described below) with 200 U of Moloney murine leukemia virus reverse transcriptase (NEB), 2 mM deoxynucleoside triphosphate, 40 μM iraD ORF specific primer (5′-TTA GCT GAC ATT CTC CAG CGT CGC ACT GCG-3′), and 1× Moloney murine leukemia virus RT buffer at 42°C for 1 h, followed by treatment with 10 μg of RNase A (Sigma)/ml and 5 U of RNase H (NEB) at 37°C for 30 min. cDNA was purified by using a QIAquick PCR purification kit (Qiagen). .. After ligation of cDNA with an anchor oligonucleotide (5′-TTT AGT GAG GGT TAA TAA GCG GCC GCG TCG TGA CTG GGA GCG C-3′), touchdown PCR was performed by using 1 U of Phusion polymerase (Finnzyme), 12 ng of purified anchored-cDNA, 0.5 μM anchor, and iraD ORF specific primers (5′-CTC CCA GTC ACG ACG CGG CCG C-3′ and 5′-CAA ACG TTA GCG GTT CAT CG-3′).

    Gel Purification:

    Article Title: The wright stuff: reimagining path analysis reveals novel components of the sex determination hierarchy in drosophila melanogaster
    Article Snippet: Second strand cDNA was synthesized using DNA polymerase I in combination with ribonuclease H (NEB). .. We then enriched for cDNA templates by performing multiplex incorporating PCR reactions (≤18 cycles), and isolating 250–550 base pair fragments by gel purification.

    Ligation:

    Article Title: Growth Phase and (p)ppGpp Control of IraD, a Regulator of RpoS Stability, in Escherichia coli ▿
    Article Snippet: The 5′ ends of iraD mRNA were mapped by using 5′ RACE (rapid amplification of cDNA ends) as previously described ( ) with the following modifications. cDNA was prepared by incubation of 2 μg of total RNA (isolated as described below) with 200 U of Moloney murine leukemia virus reverse transcriptase (NEB), 2 mM deoxynucleoside triphosphate, 40 μM iraD ORF specific primer (5′-TTA GCT GAC ATT CTC CAG CGT CGC ACT GCG-3′), and 1× Moloney murine leukemia virus RT buffer at 42°C for 1 h, followed by treatment with 10 μg of RNase A (Sigma)/ml and 5 U of RNase H (NEB) at 37°C for 30 min. cDNA was purified by using a QIAquick PCR purification kit (Qiagen). .. After ligation of cDNA with an anchor oligonucleotide (5′-TTT AGT GAG GGT TAA TAA GCG GCC GCG TCG TGA CTG GGA GCG C-3′), touchdown PCR was performed by using 1 U of Phusion polymerase (Finnzyme), 12 ng of purified anchored-cDNA, 0.5 μM anchor, and iraD ORF specific primers (5′-CTC CCA GTC ACG ACG CGG CCG C-3′ and 5′-CAA ACG TTA GCG GTT CAT CG-3′).

    Infection:

    Article Title: Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia
    Article Snippet: High-throughput sequencing was performed in parallel on random-primed cDNA preparations from purified virus and infected brain tissue RNA. .. The cDNA was RNase H treated prior to second-strand synthesis with Klenow fragment (New England Biolabs, Ipswich, MA).

    other:

    Article Title: Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA
    Article Snippet: Relative to cleavage of the unstructured target, RNase H only weakly cleaved all four sites, while DISC demonstrated more robust cleavage of TR6, TR8 and TR11 (Figure and ).

    Article Title: Strong transcription blockage mediated by R-loop formation within a G-rich homopurine–homopyrimidine sequence localized in the vicinity of the promoter
    Article Snippet: Note that in the presence of RNase H, most of the run-off transcripts have the same (i.e. full-size) length, as in the absence of RNase H (Figure , left panel, lane 4 versus lane 2), and the yields of full-size products in the presence of RNase H are similar for the promoter–proximal and promoter–distal substrates (Figure ); however, some minor fraction of shorter transcripts appears in the presence of RNase H (see the square bracket near the lane 4, Figure , right panel).

    Isolation:

    Article Title: Constitutive smooth muscle tumour necrosis factor regulates microvascular myogenic responsiveness and systemic blood pressure
    Article Snippet: Briefly, before starting the RT reaction, the isolated RNA was combined with deoxynucleotide triphosphates (dNTPs) and random heximers, heated to 65 °C for 5 min and then chilled on ice for 2 min. .. Immediately following the RT reaction, RNA was removed by adding 2.5 U RNase H (New England Biolabs) and incubating the reaction at 37 °C for 20 min; the RNAse enzyme was subsequently inactivated by heating to 65 °C for 15 min. Target genes were amplified with an i-Taq DNA polymerase kit (FroggaBio).

    Article Title: Growth Phase and (p)ppGpp Control of IraD, a Regulator of RpoS Stability, in Escherichia coli ▿
    Article Snippet: .. The 5′ ends of iraD mRNA were mapped by using 5′ RACE (rapid amplification of cDNA ends) as previously described ( ) with the following modifications. cDNA was prepared by incubation of 2 μg of total RNA (isolated as described below) with 200 U of Moloney murine leukemia virus reverse transcriptase (NEB), 2 mM deoxynucleoside triphosphate, 40 μM iraD ORF specific primer (5′-TTA GCT GAC ATT CTC CAG CGT CGC ACT GCG-3′), and 1× Moloney murine leukemia virus RT buffer at 42°C for 1 h, followed by treatment with 10 μg of RNase A (Sigma)/ml and 5 U of RNase H (NEB) at 37°C for 30 min. cDNA was purified by using a QIAquick PCR purification kit (Qiagen). .. After ligation of cDNA with an anchor oligonucleotide (5′-TTT AGT GAG GGT TAA TAA GCG GCC GCG TCG TGA CTG GGA GCG C-3′), touchdown PCR was performed by using 1 U of Phusion polymerase (Finnzyme), 12 ng of purified anchored-cDNA, 0.5 μM anchor, and iraD ORF specific primers (5′-CTC CCA GTC ACG ACG CGG CCG C-3′ and 5′-CAA ACG TTA GCG GTT CAT CG-3′).

    Sequencing:

    Article Title: Identification and Expression Analysis of Candidate Odorant-Binding Protein and Chemosensory Protein Genes by Antennal Transcriptome of Sitobion avenae
    Article Snippet: Paragraph title: Transcriptome sequencing, assembly and functional annotation ... Single-stranded (ss) cDNA was synthesized using a random hexamer primer, M-MuLV Reverse Transcriptase and DNA Polymerase I and RNase H (NEB, USA).

    Article Title: Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia
    Article Snippet: Paragraph title: Nucleic acid extraction, library preparation, and high-throughput sequencing. ... The cDNA was RNase H treated prior to second-strand synthesis with Klenow fragment (New England Biolabs, Ipswich, MA).

    Article Title: The wright stuff: reimagining path analysis reveals novel components of the sex determination hierarchy in drosophila melanogaster
    Article Snippet: Second strand cDNA was synthesized using DNA polymerase I in combination with ribonuclease H (NEB). .. Illumina sequencing adapters were then ligated to both ends of the cDNA templates using Fast-Link™ DNA Ligation Kit (Epicentre).

    Recombinant:

    Article Title: Constitutive smooth muscle tumour necrosis factor regulates microvascular myogenic responsiveness and systemic blood pressure
    Article Snippet: The remaining components required for the RT reaction were then added: each RT reaction (40 μl volume) contained 8 μl kit-provided 5 × buffer solution, 0.5 mmol l−1 dNTPs (Genedirex Inc.), 100 ng random heximers (Invitrogen Life Technologies), 5 μmol l−1 dithiothreitol, 400 U Superscript III reverse transcriptase (Invitrogen Life Technologies) and 80 U RNaseOut recombinant ribonuclease inhibitor (Invitrogen Life Technologies). .. Immediately following the RT reaction, RNA was removed by adding 2.5 U RNase H (New England Biolabs) and incubating the reaction at 37 °C for 20 min; the RNAse enzyme was subsequently inactivated by heating to 65 °C for 15 min. Target genes were amplified with an i-Taq DNA polymerase kit (FroggaBio).

    Cellular Antioxidant Activity Assay:

    Article Title: Growth Phase and (p)ppGpp Control of IraD, a Regulator of RpoS Stability, in Escherichia coli ▿
    Article Snippet: The 5′ ends of iraD mRNA were mapped by using 5′ RACE (rapid amplification of cDNA ends) as previously described ( ) with the following modifications. cDNA was prepared by incubation of 2 μg of total RNA (isolated as described below) with 200 U of Moloney murine leukemia virus reverse transcriptase (NEB), 2 mM deoxynucleoside triphosphate, 40 μM iraD ORF specific primer (5′-TTA GCT GAC ATT CTC CAG CGT CGC ACT GCG-3′), and 1× Moloney murine leukemia virus RT buffer at 42°C for 1 h, followed by treatment with 10 μg of RNase A (Sigma)/ml and 5 U of RNase H (NEB) at 37°C for 30 min. cDNA was purified by using a QIAquick PCR purification kit (Qiagen). .. PCR products were resolved on 1.5% agarose gels and bands were excised by using a QIAEX II gel extraction kit (Qiagen) and sequenced using the iraD ORF and anchor-specific primers (5′-TTC TTT CAA ATT AAC CTG CAA CGC C-3′ and 5′-TAA AAA GAG TGA GGA GAT CGC-3′, respectively).

    RNA Sequencing Assay:

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq
    Article Snippet: .. Another possibility is that the cell may target mRNAs with retained first-introns that form R-loops to be degraded by RNase H. In this model, mRNAs with retained introns that are observable by RNA-seq must have mechanisms to prevent the RNA from forming a long R-loop that would be susceptible to RNase H degradation. ..

    Fluorescence:

    Article Title: Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus
    Article Snippet: .. After 5 min, 2 μl of an enzyme mixture containing 2.6 μg of bovine serum albumin (Promega Co.), 40 international units (IU) of T7 RNA polymerase (Novagen Inc., Madison, WI), 8 IU of avian myeloblastosis virus reverse transcriptase (Seikagaku, Ijamsville, Md.), 0.2 IU of RNase H (New England Biolabs, Beverly, MA), and 12.5 IU of RNasin (Amersham Biotech) were added to each well, followed by incubation at 40° ± 1°C for 150 min. Fluorescence intensity data were recorded every minute of the NASBA reaction. .. The threshold cycle of each amplification reaction was calculated based on the first cycle at which the fluorescence was 10-fold higher than the standard deviation of the mean baseline emission.

    Magnetic Beads:

    Article Title: Identification and Expression Analysis of Candidate Odorant-Binding Protein and Chemosensory Protein Genes by Antennal Transcriptome of Sitobion avenae
    Article Snippet: After assessing RNA quality on a spectrophotometer NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA), a 3 μg RNA sample with standard quality ratios (1.8 < OD260/280 < 2.1) was purified using poly-T oligo-attached magnetic beads. .. Single-stranded (ss) cDNA was synthesized using a random hexamer primer, M-MuLV Reverse Transcriptase and DNA Polymerase I and RNase H (NEB, USA).

    Mutagenesis:

    Article Title: The wright stuff: reimagining path analysis reveals novel components of the sex determination hierarchy in drosophila melanogaster
    Article Snippet: Paragraph title: dsx null mutant for GRN validation ... Second strand cDNA was synthesized using DNA polymerase I in combination with ribonuclease H (NEB).

    Random Primed:

    Article Title: Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia
    Article Snippet: High-throughput sequencing was performed in parallel on random-primed cDNA preparations from purified virus and infected brain tissue RNA. .. The cDNA was RNase H treated prior to second-strand synthesis with Klenow fragment (New England Biolabs, Ipswich, MA).

    Polymerase Chain Reaction:

    Article Title: Identification and genomic characterization of the first isolate of bluetongue virus serotype 5 detected in Australia
    Article Snippet: Complementary DNA was reverse transcribed using the Superscript III system (Invitrogen) in the presence of 50 ng of random hexamers and 0.5 ng of primers specific to the 5’ and 3’ ends common to all BTV genome segments (5’ = GTTAAAN =3’ = and 5’ = GTAAGTN = 3’ = respectively) (Mertens & Sangar ) and digested with RNase H (New England Biolabs). .. The cDNA was purified using a MiniElute PCR Purification Kit (Qiagen) and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), prior to library preparation for next generation sequencing.

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning
    Article Snippet: Then, DNA–RNA hybrids were digested with 5 U of RNAse H (NEB) for 15 min at 37°C, extracted with phenol and ethanol precipitated. .. Finally, the resulting cDNA was amplified by PCR using oligo(dT) and oligo 9 primers, and sequenced.

    Article Title: Chlorhexidine Induces VanA-Type Vancomycin Resistance Genes in Enterococci
    Article Snippet: .. RNase H (NEB) was added to remove RNA, and cDNA was purified using the QIAquick PCR purification kit (Qiagen). .. Five nanograms of cDNA was used as the template in quantitative reverse transcription-PCR (RT-qPCR) with primers to amplify internal regions of vanA , vanB , or clpX (see Table S1 in the supplemental material).

    Article Title: Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus
    Article Snippet: Five microliters of purified viral RNA was added to the 18 μl of pre-reaction mixture in a 0.5-ml Bio-Rad PCR plate, which was incubated for 5 min at 65°C in order to disrupt any secondary structure in the target RNA. .. After 5 min, 2 μl of an enzyme mixture containing 2.6 μg of bovine serum albumin (Promega Co.), 40 international units (IU) of T7 RNA polymerase (Novagen Inc., Madison, WI), 8 IU of avian myeloblastosis virus reverse transcriptase (Seikagaku, Ijamsville, Md.), 0.2 IU of RNase H (New England Biolabs, Beverly, MA), and 12.5 IU of RNasin (Amersham Biotech) were added to each well, followed by incubation at 40° ± 1°C for 150 min. Fluorescence intensity data were recorded every minute of the NASBA reaction.

    Article Title: Identification and Expression Analysis of Candidate Odorant-Binding Protein and Chemosensory Protein Genes by Antennal Transcriptome of Sitobion avenae
    Article Snippet: Single-stranded (ss) cDNA was synthesized using a random hexamer primer, M-MuLV Reverse Transcriptase and DNA Polymerase I and RNase H (NEB, USA). .. Then 3 μL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C prior to PCR.

    Article Title: Growth Phase and (p)ppGpp Control of IraD, a Regulator of RpoS Stability, in Escherichia coli ▿
    Article Snippet: .. The 5′ ends of iraD mRNA were mapped by using 5′ RACE (rapid amplification of cDNA ends) as previously described ( ) with the following modifications. cDNA was prepared by incubation of 2 μg of total RNA (isolated as described below) with 200 U of Moloney murine leukemia virus reverse transcriptase (NEB), 2 mM deoxynucleoside triphosphate, 40 μM iraD ORF specific primer (5′-TTA GCT GAC ATT CTC CAG CGT CGC ACT GCG-3′), and 1× Moloney murine leukemia virus RT buffer at 42°C for 1 h, followed by treatment with 10 μg of RNase A (Sigma)/ml and 5 U of RNase H (NEB) at 37°C for 30 min. cDNA was purified by using a QIAquick PCR purification kit (Qiagen). .. After ligation of cDNA with an anchor oligonucleotide (5′-TTT AGT GAG GGT TAA TAA GCG GCC GCG TCG TGA CTG GGA GCG C-3′), touchdown PCR was performed by using 1 U of Phusion polymerase (Finnzyme), 12 ng of purified anchored-cDNA, 0.5 μM anchor, and iraD ORF specific primers (5′-CTC CCA GTC ACG ACG CGG CCG C-3′ and 5′-CAA ACG TTA GCG GTT CAT CG-3′).

    Article Title: Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia
    Article Snippet: The cDNA was RNase H treated prior to second-strand synthesis with Klenow fragment (New England Biolabs, Ipswich, MA). .. Sheared product was purified (AxyPrep Mag PCR cleanup beads; Axygen/Corning, Corning, NY), and libraries were constructed using KAPA library preparation kits (KAPA, Wilmington, MA) with 6-nt bar code adapters.

    Article Title: The wright stuff: reimagining path analysis reveals novel components of the sex determination hierarchy in drosophila melanogaster
    Article Snippet: Second strand cDNA was synthesized using DNA polymerase I in combination with ribonuclease H (NEB). .. We then enriched for cDNA templates by performing multiplex incorporating PCR reactions (≤18 cycles), and isolating 250–550 base pair fragments by gel purification.

    Functional Assay:

    Article Title: Identification and Expression Analysis of Candidate Odorant-Binding Protein and Chemosensory Protein Genes by Antennal Transcriptome of Sitobion avenae
    Article Snippet: Paragraph title: Transcriptome sequencing, assembly and functional annotation ... Single-stranded (ss) cDNA was synthesized using a random hexamer primer, M-MuLV Reverse Transcriptase and DNA Polymerase I and RNase H (NEB, USA).

    Purification:

    Article Title: Identification and genomic characterization of the first isolate of bluetongue virus serotype 5 detected in Australia
    Article Snippet: Complementary DNA was reverse transcribed using the Superscript III system (Invitrogen) in the presence of 50 ng of random hexamers and 0.5 ng of primers specific to the 5’ and 3’ ends common to all BTV genome segments (5’ = GTTAAAN =3’ = and 5’ = GTAAGTN = 3’ = respectively) (Mertens & Sangar ) and digested with RNase H (New England Biolabs). .. The cDNA was purified using a MiniElute PCR Purification Kit (Qiagen) and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), prior to library preparation for next generation sequencing.

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning
    Article Snippet: Mapping of 3′ oligo 4–18S rRNA pairing 5 pmol of oligo 4 or VIC–oligo 4 were incubated with 30 pmol of 40S subunits purified from RRL and incubated for 30 min at 30°C in polysome buffer. .. Then, DNA–RNA hybrids were digested with 5 U of RNAse H (NEB) for 15 min at 37°C, extracted with phenol and ethanol precipitated.

    Article Title: Chlorhexidine Induces VanA-Type Vancomycin Resistance Genes in Enterococci
    Article Snippet: .. RNase H (NEB) was added to remove RNA, and cDNA was purified using the QIAquick PCR purification kit (Qiagen). .. Five nanograms of cDNA was used as the template in quantitative reverse transcription-PCR (RT-qPCR) with primers to amplify internal regions of vanA , vanB , or clpX (see Table S1 in the supplemental material).

    Article Title: Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus
    Article Snippet: Five microliters of purified viral RNA was added to the 18 μl of pre-reaction mixture in a 0.5-ml Bio-Rad PCR plate, which was incubated for 5 min at 65°C in order to disrupt any secondary structure in the target RNA. .. After 5 min, 2 μl of an enzyme mixture containing 2.6 μg of bovine serum albumin (Promega Co.), 40 international units (IU) of T7 RNA polymerase (Novagen Inc., Madison, WI), 8 IU of avian myeloblastosis virus reverse transcriptase (Seikagaku, Ijamsville, Md.), 0.2 IU of RNase H (New England Biolabs, Beverly, MA), and 12.5 IU of RNasin (Amersham Biotech) were added to each well, followed by incubation at 40° ± 1°C for 150 min. Fluorescence intensity data were recorded every minute of the NASBA reaction.

    Article Title: Identification and Expression Analysis of Candidate Odorant-Binding Protein and Chemosensory Protein Genes by Antennal Transcriptome of Sitobion avenae
    Article Snippet: After assessing RNA quality on a spectrophotometer NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA), a 3 μg RNA sample with standard quality ratios (1.8 < OD260/280 < 2.1) was purified using poly-T oligo-attached magnetic beads. .. Single-stranded (ss) cDNA was synthesized using a random hexamer primer, M-MuLV Reverse Transcriptase and DNA Polymerase I and RNase H (NEB, USA).

    Article Title: Growth Phase and (p)ppGpp Control of IraD, a Regulator of RpoS Stability, in Escherichia coli ▿
    Article Snippet: .. The 5′ ends of iraD mRNA were mapped by using 5′ RACE (rapid amplification of cDNA ends) as previously described ( ) with the following modifications. cDNA was prepared by incubation of 2 μg of total RNA (isolated as described below) with 200 U of Moloney murine leukemia virus reverse transcriptase (NEB), 2 mM deoxynucleoside triphosphate, 40 μM iraD ORF specific primer (5′-TTA GCT GAC ATT CTC CAG CGT CGC ACT GCG-3′), and 1× Moloney murine leukemia virus RT buffer at 42°C for 1 h, followed by treatment with 10 μg of RNase A (Sigma)/ml and 5 U of RNase H (NEB) at 37°C for 30 min. cDNA was purified by using a QIAquick PCR purification kit (Qiagen). .. After ligation of cDNA with an anchor oligonucleotide (5′-TTT AGT GAG GGT TAA TAA GCG GCC GCG TCG TGA CTG GGA GCG C-3′), touchdown PCR was performed by using 1 U of Phusion polymerase (Finnzyme), 12 ng of purified anchored-cDNA, 0.5 μM anchor, and iraD ORF specific primers (5′-CTC CCA GTC ACG ACG CGG CCG C-3′ and 5′-CAA ACG TTA GCG GTT CAT CG-3′).

    Article Title: Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia
    Article Snippet: Sequencing from purified virus on the Illumina HiSeq 2500 platform (Illumina) resulted in an average of ~200 million reads (100 nucleotides [nt]) per sample. .. The cDNA was RNase H treated prior to second-strand synthesis with Klenow fragment (New England Biolabs, Ipswich, MA).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Constitutive smooth muscle tumour necrosis factor regulates microvascular myogenic responsiveness and systemic blood pressure
    Article Snippet: Paragraph title: Reverse transcription—polymerase chain reaction ... Immediately following the RT reaction, RNA was removed by adding 2.5 U RNase H (New England Biolabs) and incubating the reaction at 37 °C for 20 min; the RNAse enzyme was subsequently inactivated by heating to 65 °C for 15 min. Target genes were amplified with an i-Taq DNA polymerase kit (FroggaBio).

    Construct:

    Article Title: Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia
    Article Snippet: The cDNA was RNase H treated prior to second-strand synthesis with Klenow fragment (New England Biolabs, Ipswich, MA). .. Sheared product was purified (AxyPrep Mag PCR cleanup beads; Axygen/Corning, Corning, NY), and libraries were constructed using KAPA library preparation kits (KAPA, Wilmington, MA) with 6-nt bar code adapters.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Growth Phase and (p)ppGpp Control of IraD, a Regulator of RpoS Stability, in Escherichia coli ▿
    Article Snippet: The 5′ ends of iraD mRNA were mapped by using 5′ RACE (rapid amplification of cDNA ends) as previously described ( ) with the following modifications. cDNA was prepared by incubation of 2 μg of total RNA (isolated as described below) with 200 U of Moloney murine leukemia virus reverse transcriptase (NEB), 2 mM deoxynucleoside triphosphate, 40 μM iraD ORF specific primer (5′-TTA GCT GAC ATT CTC CAG CGT CGC ACT GCG-3′), and 1× Moloney murine leukemia virus RT buffer at 42°C for 1 h, followed by treatment with 10 μg of RNase A (Sigma)/ml and 5 U of RNase H (NEB) at 37°C for 30 min. cDNA was purified by using a QIAquick PCR purification kit (Qiagen). .. After ligation of cDNA with an anchor oligonucleotide (5′-TTT AGT GAG GGT TAA TAA GCG GCC GCG TCG TGA CTG GGA GCG C-3′), touchdown PCR was performed by using 1 U of Phusion polymerase (Finnzyme), 12 ng of purified anchored-cDNA, 0.5 μM anchor, and iraD ORF specific primers (5′-CTC CCA GTC ACG ACG CGG CCG C-3′ and 5′-CAA ACG TTA GCG GTT CAT CG-3′).

    Rapid Amplification of cDNA Ends:

    Article Title: Growth Phase and (p)ppGpp Control of IraD, a Regulator of RpoS Stability, in Escherichia coli ▿
    Article Snippet: .. The 5′ ends of iraD mRNA were mapped by using 5′ RACE (rapid amplification of cDNA ends) as previously described ( ) with the following modifications. cDNA was prepared by incubation of 2 μg of total RNA (isolated as described below) with 200 U of Moloney murine leukemia virus reverse transcriptase (NEB), 2 mM deoxynucleoside triphosphate, 40 μM iraD ORF specific primer (5′-TTA GCT GAC ATT CTC CAG CGT CGC ACT GCG-3′), and 1× Moloney murine leukemia virus RT buffer at 42°C for 1 h, followed by treatment with 10 μg of RNase A (Sigma)/ml and 5 U of RNase H (NEB) at 37°C for 30 min. cDNA was purified by using a QIAquick PCR purification kit (Qiagen). .. After ligation of cDNA with an anchor oligonucleotide (5′-TTT AGT GAG GGT TAA TAA GCG GCC GCG TCG TGA CTG GGA GCG C-3′), touchdown PCR was performed by using 1 U of Phusion polymerase (Finnzyme), 12 ng of purified anchored-cDNA, 0.5 μM anchor, and iraD ORF specific primers (5′-CTC CCA GTC ACG ACG CGG CCG C-3′ and 5′-CAA ACG TTA GCG GTT CAT CG-3′).

    Hybridization:

    Article Title: Identification and Expression Analysis of Candidate Odorant-Binding Protein and Chemosensory Protein Genes by Antennal Transcriptome of Sitobion avenae
    Article Snippet: Single-stranded (ss) cDNA was synthesized using a random hexamer primer, M-MuLV Reverse Transcriptase and DNA Polymerase I and RNase H (NEB, USA). .. The 3' ends of the DNA fragments were adenylated and the NEBNext Adaptor was ligated to the fragments for hybridization.

    Real-time Polymerase Chain Reaction:

    Article Title: Mechanical Genomic Studies Reveal the Role of d-Alanine Metabolism in Pseudomonas aeruginosa Cell Stiffness
    Article Snippet: Paragraph title: Quantitative PCR. ... Newly synthesized cDNA was treated with New England Biolabs RNase H to digest RNA hybridized to cDNA.

    Article Title: Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus
    Article Snippet: A Bio-Rad (Hercules, CA) icycler iQ Real-Time PCR Detection System was used to perform the real-time NASBA assay. .. After 5 min, 2 μl of an enzyme mixture containing 2.6 μg of bovine serum albumin (Promega Co.), 40 international units (IU) of T7 RNA polymerase (Novagen Inc., Madison, WI), 8 IU of avian myeloblastosis virus reverse transcriptase (Seikagaku, Ijamsville, Md.), 0.2 IU of RNase H (New England Biolabs, Beverly, MA), and 12.5 IU of RNasin (Amersham Biotech) were added to each well, followed by incubation at 40° ± 1°C for 150 min. Fluorescence intensity data were recorded every minute of the NASBA reaction.

    Multiplex Assay:

    Article Title: The wright stuff: reimagining path analysis reveals novel components of the sex determination hierarchy in drosophila melanogaster
    Article Snippet: Second strand cDNA was synthesized using DNA polymerase I in combination with ribonuclease H (NEB). .. We then enriched for cDNA templates by performing multiplex incorporating PCR reactions (≤18 cycles), and isolating 250–550 base pair fragments by gel purification.

    Next-Generation Sequencing:

    Article Title: Identification and genomic characterization of the first isolate of bluetongue virus serotype 5 detected in Australia
    Article Snippet: Complementary DNA was reverse transcribed using the Superscript III system (Invitrogen) in the presence of 50 ng of random hexamers and 0.5 ng of primers specific to the 5’ and 3’ ends common to all BTV genome segments (5’ = GTTAAAN =3’ = and 5’ = GTAAGTN = 3’ = respectively) (Mertens & Sangar ) and digested with RNase H (New England Biolabs). .. The cDNA was purified using a MiniElute PCR Purification Kit (Qiagen) and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), prior to library preparation for next generation sequencing.

    Incubation:

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning
    Article Snippet: Mapping of 3′ oligo 4–18S rRNA pairing 5 pmol of oligo 4 or VIC–oligo 4 were incubated with 30 pmol of 40S subunits purified from RRL and incubated for 30 min at 30°C in polysome buffer. .. Then, DNA–RNA hybrids were digested with 5 U of RNAse H (NEB) for 15 min at 37°C, extracted with phenol and ethanol precipitated.

    Article Title: Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus
    Article Snippet: .. After 5 min, 2 μl of an enzyme mixture containing 2.6 μg of bovine serum albumin (Promega Co.), 40 international units (IU) of T7 RNA polymerase (Novagen Inc., Madison, WI), 8 IU of avian myeloblastosis virus reverse transcriptase (Seikagaku, Ijamsville, Md.), 0.2 IU of RNase H (New England Biolabs, Beverly, MA), and 12.5 IU of RNasin (Amersham Biotech) were added to each well, followed by incubation at 40° ± 1°C for 150 min. Fluorescence intensity data were recorded every minute of the NASBA reaction. .. The threshold cycle of each amplification reaction was calculated based on the first cycle at which the fluorescence was 10-fold higher than the standard deviation of the mean baseline emission.

    Article Title: Constitutive smooth muscle tumour necrosis factor regulates microvascular myogenic responsiveness and systemic blood pressure
    Article Snippet: Each RT reaction was incubated at 25 °C for 5 min and then 50 °C for 1 h; the Superscript enzyme was then inactivated by heating to 70 °C for 15 min. .. Immediately following the RT reaction, RNA was removed by adding 2.5 U RNase H (New England Biolabs) and incubating the reaction at 37 °C for 20 min; the RNAse enzyme was subsequently inactivated by heating to 65 °C for 15 min. Target genes were amplified with an i-Taq DNA polymerase kit (FroggaBio).

    Article Title: Growth Phase and (p)ppGpp Control of IraD, a Regulator of RpoS Stability, in Escherichia coli ▿
    Article Snippet: .. The 5′ ends of iraD mRNA were mapped by using 5′ RACE (rapid amplification of cDNA ends) as previously described ( ) with the following modifications. cDNA was prepared by incubation of 2 μg of total RNA (isolated as described below) with 200 U of Moloney murine leukemia virus reverse transcriptase (NEB), 2 mM deoxynucleoside triphosphate, 40 μM iraD ORF specific primer (5′-TTA GCT GAC ATT CTC CAG CGT CGC ACT GCG-3′), and 1× Moloney murine leukemia virus RT buffer at 42°C for 1 h, followed by treatment with 10 μg of RNase A (Sigma)/ml and 5 U of RNase H (NEB) at 37°C for 30 min. cDNA was purified by using a QIAquick PCR purification kit (Qiagen). .. After ligation of cDNA with an anchor oligonucleotide (5′-TTT AGT GAG GGT TAA TAA GCG GCC GCG TCG TGA CTG GGA GCG C-3′), touchdown PCR was performed by using 1 U of Phusion polymerase (Finnzyme), 12 ng of purified anchored-cDNA, 0.5 μM anchor, and iraD ORF specific primers (5′-CTC CCA GTC ACG ACG CGG CCG C-3′ and 5′-CAA ACG TTA GCG GTT CAT CG-3′).

    Spectrophotometry:

    Article Title: Identification and Expression Analysis of Candidate Odorant-Binding Protein and Chemosensory Protein Genes by Antennal Transcriptome of Sitobion avenae
    Article Snippet: After assessing RNA quality on a spectrophotometer NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA), a 3 μg RNA sample with standard quality ratios (1.8 < OD260/280 < 2.1) was purified using poly-T oligo-attached magnetic beads. .. Single-stranded (ss) cDNA was synthesized using a random hexamer primer, M-MuLV Reverse Transcriptase and DNA Polymerase I and RNase H (NEB, USA).

    Concentration Assay:

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq
    Article Snippet: .. In each of these final two wash steps, RNase H was added to a final concentration of 0.5 U/µl to the ‘RNase H’ treated sample, while dithiothreitol was added to a final concentration of 50 µM to the matched control sample. ..

    Article Title: Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus
    Article Snippet: An 18-μl pre-reaction mixture was prepared to give a final concentration in 25 μl of 40 mM Tris-HCl (pH 8.5), 50 mM KCl, 12 mM MgCl2 , 1.0 mM (each) deoxyribonucleoside triphosphate, 2.0 mM (each) ribonucleotide-5′-triphosphate, 2.0 mM dithiothreitol, 15% (vol/vol) dimethylsulfoxide, 0.2 μM of each cartridge-purified primer, and 100 nM of HAV MB. .. After 5 min, 2 μl of an enzyme mixture containing 2.6 μg of bovine serum albumin (Promega Co.), 40 international units (IU) of T7 RNA polymerase (Novagen Inc., Madison, WI), 8 IU of avian myeloblastosis virus reverse transcriptase (Seikagaku, Ijamsville, Md.), 0.2 IU of RNase H (New England Biolabs, Beverly, MA), and 12.5 IU of RNasin (Amersham Biotech) were added to each well, followed by incubation at 40° ± 1°C for 150 min. Fluorescence intensity data were recorded every minute of the NASBA reaction.

    CTG Assay:

    Article Title: Growth Phase and (p)ppGpp Control of IraD, a Regulator of RpoS Stability, in Escherichia coli ▿
    Article Snippet: The 5′ ends of iraD mRNA were mapped by using 5′ RACE (rapid amplification of cDNA ends) as previously described ( ) with the following modifications. cDNA was prepared by incubation of 2 μg of total RNA (isolated as described below) with 200 U of Moloney murine leukemia virus reverse transcriptase (NEB), 2 mM deoxynucleoside triphosphate, 40 μM iraD ORF specific primer (5′-TTA GCT GAC ATT CTC CAG CGT CGC ACT GCG-3′), and 1× Moloney murine leukemia virus RT buffer at 42°C for 1 h, followed by treatment with 10 μg of RNase A (Sigma)/ml and 5 U of RNase H (NEB) at 37°C for 30 min. cDNA was purified by using a QIAquick PCR purification kit (Qiagen). .. After ligation of cDNA with an anchor oligonucleotide (5′-TTT AGT GAG GGT TAA TAA GCG GCC GCG TCG TGA CTG GGA GCG C-3′), touchdown PCR was performed by using 1 U of Phusion polymerase (Finnzyme), 12 ng of purified anchored-cDNA, 0.5 μM anchor, and iraD ORF specific primers (5′-CTC CCA GTC ACG ACG CGG CCG C-3′ and 5′-CAA ACG TTA GCG GTT CAT CG-3′).

    High Throughput Screening Assay:

    Article Title: Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia
    Article Snippet: Paragraph title: Nucleic acid extraction, library preparation, and high-throughput sequencing. ... The cDNA was RNase H treated prior to second-strand synthesis with Klenow fragment (New England Biolabs, Ipswich, MA).

    Gel Extraction:

    Article Title: Growth Phase and (p)ppGpp Control of IraD, a Regulator of RpoS Stability, in Escherichia coli ▿
    Article Snippet: The 5′ ends of iraD mRNA were mapped by using 5′ RACE (rapid amplification of cDNA ends) as previously described ( ) with the following modifications. cDNA was prepared by incubation of 2 μg of total RNA (isolated as described below) with 200 U of Moloney murine leukemia virus reverse transcriptase (NEB), 2 mM deoxynucleoside triphosphate, 40 μM iraD ORF specific primer (5′-TTA GCT GAC ATT CTC CAG CGT CGC ACT GCG-3′), and 1× Moloney murine leukemia virus RT buffer at 42°C for 1 h, followed by treatment with 10 μg of RNase A (Sigma)/ml and 5 U of RNase H (NEB) at 37°C for 30 min. cDNA was purified by using a QIAquick PCR purification kit (Qiagen). .. PCR products were resolved on 1.5% agarose gels and bands were excised by using a QIAEX II gel extraction kit (Qiagen) and sequenced using the iraD ORF and anchor-specific primers (5′-TTC TTT CAA ATT AAC CTG CAA CGC C-3′ and 5′-TAA AAA GAG TGA GGA GAT CGC-3′, respectively).

    Fluorescence In Situ Hybridization:

    Article Title: Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia
    Article Snippet: For library preparations, total RNA was extracted from purified virus particles and infected fish brain tissue samples with TRI reagent (Sigma-Aldrich, St. Louis, MO). .. The cDNA was RNase H treated prior to second-strand synthesis with Klenow fragment (New England Biolabs, Ipswich, MA).

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    New England Biolabs rnase h pre reactions
    Schematic representation of yeast in vivo RNA–protein Ni 2+ -pull down (RaP-NiP) assay using formaldehyde crosslinking. The basic scheme of the RaP-NiP is described in the form of a flowchart. Green and red balls represent 40S ribosomes and eIF3 complexes, respectively, grey balls stand for the Ni 2+ beads, and purple and blue balls depict some non-specific RNA binding proteins. Exponentially growing yeast cells were crosslinked with 1% formaldehyde. Crosslinking was stopped by adding glycine and the fixed cells were lysed using glass beads by rigorous vortexing. Pre-cleared whole cell extract (WCE) containing RaP-NiP mRNAs in protein-RNA complexes were selectively digested with <t>RNase</t> H using sequence specific custom-made oligos. The resulting specific mRNA segments were purified with the help of the His-tagged a/TIF32 subunit of yeast eIF3 or its mutant variants using the Ni-NTA sepharose beads. Thus isolated protein-RNA complexes were subsequently treated with Proteinase K, and the captured RNAs were further purified by hot phenol extraction, reverse transcribed and their amounts were then quantified by qRT-PCR. The schematic boxed on the right-hand side illustrates typical amounts of RNAse H digested RNA segments of REI-permissive uORF1 and REI-non-permissive uORF4 from the GCN4 mRNA leader co-purifying with eIF3, the typical ratio of which is ∼4:1.
    Rnase H Pre Reactions, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase h pre reactions/product/New England Biolabs
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rnase h pre reactions - by Bioz Stars, 2020-02
    95/100 stars
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    95
    New England Biolabs rnase h
    R‐loops promote ( GAA ) 10 ‐dependent epigenetic instability of  BU ‐1 DRIP‐qPCR analysis reveals accumulation of R‐loops across the  BU‐1  locus in  primpol  cells. The DRIP signal was calculated as enrichment over RNase H‐treated samples and was normalised to −0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired  t ‐test was used to compare differences between matched amplicons in  primpol BU‐1A (GAA)10  and the other cell lines indicated. **** P  ≤ 0.0001, ns = not significant. DNA:RNA hybrids in  primpol BU‐1A (GAA)10 :Gg RNase H1 (see also   Appendix Fig S5 ) and  primpol BU‐1A (GAA)10 :hPrimPol. An unpaired  t ‐test on three biological replicates was used to compare differences to  primpol BU‐1A (GAA)10  for each matched amplicon. The bar represents the mean, and whiskers represent the SD. *** P  ≤ 0.001, ** P  ≤ 0.01, ns = not significant. Overexpression of chicken RNase H1 prevents (GAA) 10 ‐induced  BU‐1A  epigenetic instability in  primpol  cells. Fluctuation analysis was performed on three  primpol BU‐1A (GAA)10  clones. One‐way ANOVA was used to calculate the significance of differences in  BU‐1  instability between  primpol BU‐1A ΔG4  and other cell lines. **** P  ≤ 0.0001, ns = not significant. Diagram of the RNase H1 hybrid binding domain (HBD)–mCherry fusion and flow cytometry expression profiles of the construct in four clones. Western blots of the same four clones are shown in   Appendix Fig S6 . R‐loop stabilisation induces epigenetic instability of  BU‐1 . Bu‐1a fluctuation analysis of wild‐type cells expressing HBD‐mCherry. The scatter plots pool results from at least two different clones with matched HBD expression. Mean ± SD reported. **** P  ≤ 0.0001, *** P  ≤ 0.001, ns = not significant; one‐way ANOVA.
    Rnase H, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase h/product/New England Biolabs
    Average 95 stars, based on 228 article reviews
    Price from $9.99 to $1999.99
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    Schematic representation of yeast in vivo RNA–protein Ni 2+ -pull down (RaP-NiP) assay using formaldehyde crosslinking. The basic scheme of the RaP-NiP is described in the form of a flowchart. Green and red balls represent 40S ribosomes and eIF3 complexes, respectively, grey balls stand for the Ni 2+ beads, and purple and blue balls depict some non-specific RNA binding proteins. Exponentially growing yeast cells were crosslinked with 1% formaldehyde. Crosslinking was stopped by adding glycine and the fixed cells were lysed using glass beads by rigorous vortexing. Pre-cleared whole cell extract (WCE) containing RaP-NiP mRNAs in protein-RNA complexes were selectively digested with RNase H using sequence specific custom-made oligos. The resulting specific mRNA segments were purified with the help of the His-tagged a/TIF32 subunit of yeast eIF3 or its mutant variants using the Ni-NTA sepharose beads. Thus isolated protein-RNA complexes were subsequently treated with Proteinase K, and the captured RNAs were further purified by hot phenol extraction, reverse transcribed and their amounts were then quantified by qRT-PCR. The schematic boxed on the right-hand side illustrates typical amounts of RNAse H digested RNA segments of REI-permissive uORF1 and REI-non-permissive uORF4 from the GCN4 mRNA leader co-purifying with eIF3, the typical ratio of which is ∼4:1.

    Journal: Nucleic Acids Research

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation

    doi: 10.1093/nar/gkx049

    Figure Lengend Snippet: Schematic representation of yeast in vivo RNA–protein Ni 2+ -pull down (RaP-NiP) assay using formaldehyde crosslinking. The basic scheme of the RaP-NiP is described in the form of a flowchart. Green and red balls represent 40S ribosomes and eIF3 complexes, respectively, grey balls stand for the Ni 2+ beads, and purple and blue balls depict some non-specific RNA binding proteins. Exponentially growing yeast cells were crosslinked with 1% formaldehyde. Crosslinking was stopped by adding glycine and the fixed cells were lysed using glass beads by rigorous vortexing. Pre-cleared whole cell extract (WCE) containing RaP-NiP mRNAs in protein-RNA complexes were selectively digested with RNase H using sequence specific custom-made oligos. The resulting specific mRNA segments were purified with the help of the His-tagged a/TIF32 subunit of yeast eIF3 or its mutant variants using the Ni-NTA sepharose beads. Thus isolated protein-RNA complexes were subsequently treated with Proteinase K, and the captured RNAs were further purified by hot phenol extraction, reverse transcribed and their amounts were then quantified by qRT-PCR. The schematic boxed on the right-hand side illustrates typical amounts of RNAse H digested RNA segments of REI-permissive uORF1 and REI-non-permissive uORF4 from the GCN4 mRNA leader co-purifying with eIF3, the typical ratio of which is ∼4:1.

    Article Snippet: The RNase H pre-reactions were then left at the room temperature for 10 min, after which 10 U of RNase H (NE Biolabs) was added for 20 min at 37°C.

    Techniques: In Vivo, RNA Binding Assay, Sequencing, Purification, Mutagenesis, Isolation, Quantitative RT-PCR

    eIF3 stabilizes the post-termination 40S complexes on stop codons of REI-permissive uORF1 and uORF2 from the GCN4 mRNA leader. ( A ) A schematic showing the wild type mRNA leader of the GCN4-lacZ fusion with colored bars indicating positions of individual RPEs of uORF1, as well as of uORF2 (color coding of all four uORFs reflects their REI-permissiveness (green) or -non-permissiveness (red)—for details see Supplementary Figures S1 and S2 ). The mRNA leader was divided into several segments (X, Y n and Z), where X and Z are present in all constructs shown in panel B and contain the RNase H cutting sites (indicated by scissors) and qRT-PCR primer binding sites (indicated by red arrows) at their 5΄ and 3΄ ends, respectively. The segment X is 151 bp in length (from position –229 to –79 relative to the uORF1 AUG start codon) and the segment Z encompasses the entire downstream sequence immediately following the uORF4 stop codon (i.e. from position +223 relative to the uORF1 AUG start codon downstream). The coordinates of all Y segments, by which the constructs in panel B differ and that are in each of them placed between the X and Z segments, are given at the top or bottom of the schematic. ( B ) Schematics showing individual uORF1–4 RaP-NiP constructs with corresponding Yn inserts of the same length for uORF1-only, 3-only and 4-only constructs (top three), and for the uORF2-only and uORF4_2-only constructs (bottom two). Black bars labeled as Y3’ represent composite 13+5 nt taken from the uORF3 3΄ UTR that were placed immediately behind the first 7 nt of the uORF4 3΄ UTR (i.e. behind the Y4 segment that ends exactly at the seventh nt of the uORF4 3΄ UTR) to keep the length of the uORF4-specific Y segment the same as that of uORF1. (We could not take the entire 3΄ UTR of uORF4 because in our set-up it is an integral part of the Z segment where the downstream qPCR primer base-pairs). ( C and D ) The RNase H-cleaved uORF1 segment specifically co-purifies with the His-tagged a/TIF32 subunit of eIF3 using the in vivo RaP-NiP. (C) The YMP1 ( gcn4Δ TIF32-His ) strain was introduced either with the uORF1-only RaP-NiP construct shown in panel B or an empty vector and the resulting transformants were pre-cultured in minimal media overnight, diluted to OD 600 ∼ 0.1 and further cultivated to OD 600 ∼1. The exponentially growing cells were then subjected to RaP-NiP as described in Materials and Methods and outlined in Figure 1 . Relative qPCR product levels (in %) of the Y1 segment of uORF1 recovered from each strain with standard deviations obtained from at least three independent experiments from three independent transformants (i.e. biological replicates) normalized to reference ACT1 mRNA as well as to total RNA levels are given with the values of uORF1-only set to 100 (asterisks indicate that P

    Journal: Nucleic Acids Research

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation

    doi: 10.1093/nar/gkx049

    Figure Lengend Snippet: eIF3 stabilizes the post-termination 40S complexes on stop codons of REI-permissive uORF1 and uORF2 from the GCN4 mRNA leader. ( A ) A schematic showing the wild type mRNA leader of the GCN4-lacZ fusion with colored bars indicating positions of individual RPEs of uORF1, as well as of uORF2 (color coding of all four uORFs reflects their REI-permissiveness (green) or -non-permissiveness (red)—for details see Supplementary Figures S1 and S2 ). The mRNA leader was divided into several segments (X, Y n and Z), where X and Z are present in all constructs shown in panel B and contain the RNase H cutting sites (indicated by scissors) and qRT-PCR primer binding sites (indicated by red arrows) at their 5΄ and 3΄ ends, respectively. The segment X is 151 bp in length (from position –229 to –79 relative to the uORF1 AUG start codon) and the segment Z encompasses the entire downstream sequence immediately following the uORF4 stop codon (i.e. from position +223 relative to the uORF1 AUG start codon downstream). The coordinates of all Y segments, by which the constructs in panel B differ and that are in each of them placed between the X and Z segments, are given at the top or bottom of the schematic. ( B ) Schematics showing individual uORF1–4 RaP-NiP constructs with corresponding Yn inserts of the same length for uORF1-only, 3-only and 4-only constructs (top three), and for the uORF2-only and uORF4_2-only constructs (bottom two). Black bars labeled as Y3’ represent composite 13+5 nt taken from the uORF3 3΄ UTR that were placed immediately behind the first 7 nt of the uORF4 3΄ UTR (i.e. behind the Y4 segment that ends exactly at the seventh nt of the uORF4 3΄ UTR) to keep the length of the uORF4-specific Y segment the same as that of uORF1. (We could not take the entire 3΄ UTR of uORF4 because in our set-up it is an integral part of the Z segment where the downstream qPCR primer base-pairs). ( C and D ) The RNase H-cleaved uORF1 segment specifically co-purifies with the His-tagged a/TIF32 subunit of eIF3 using the in vivo RaP-NiP. (C) The YMP1 ( gcn4Δ TIF32-His ) strain was introduced either with the uORF1-only RaP-NiP construct shown in panel B or an empty vector and the resulting transformants were pre-cultured in minimal media overnight, diluted to OD 600 ∼ 0.1 and further cultivated to OD 600 ∼1. The exponentially growing cells were then subjected to RaP-NiP as described in Materials and Methods and outlined in Figure 1 . Relative qPCR product levels (in %) of the Y1 segment of uORF1 recovered from each strain with standard deviations obtained from at least three independent experiments from three independent transformants (i.e. biological replicates) normalized to reference ACT1 mRNA as well as to total RNA levels are given with the values of uORF1-only set to 100 (asterisks indicate that P

    Article Snippet: The RNase H pre-reactions were then left at the room temperature for 10 min, after which 10 U of RNase H (NE Biolabs) was added for 20 min at 37°C.

    Techniques: Construct, Quantitative RT-PCR, Binding Assay, Sequencing, Labeling, Real-time Polymerase Chain Reaction, In Vivo, Plasmid Preparation, Cell Culture

    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: For experiments with the RNase H treatment control, washed beads were re‐suspended in 300 μl of RNase H reaction buffer containing (NEB, M0297L) containing 4% glycerol and 20 mg/ml BSA.

    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: For experiments with the RNase H treatment control, washed beads were re‐suspended in 300 μl of RNase H reaction buffer containing (NEB, M0297L) containing 4% glycerol and 20 mg/ml BSA.

    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: For experiments with the RNase H treatment control, washed beads were re‐suspended in 300 μl of RNase H reaction buffer containing (NEB, M0297L) containing 4% glycerol and 20 mg/ml BSA.

    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: For experiments with the RNase H treatment control, washed beads were re‐suspended in 300 μl of RNase H reaction buffer containing (NEB, M0297L) containing 4% glycerol and 20 mg/ml BSA.

    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: For experiments with the RNase H treatment control, washed beads were re‐suspended in 300 μl of RNase H reaction buffer containing (NEB, M0297L) containing 4% glycerol and 20 mg/ml BSA.

    Techniques: Dissection

    R‐loops promote ( GAA ) 10 ‐dependent epigenetic instability of  BU ‐1 DRIP‐qPCR analysis reveals accumulation of R‐loops across the  BU‐1  locus in  primpol  cells. The DRIP signal was calculated as enrichment over RNase H‐treated samples and was normalised to −0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired  t ‐test was used to compare differences between matched amplicons in  primpol BU‐1A (GAA)10  and the other cell lines indicated. **** P  ≤ 0.0001, ns = not significant. DNA:RNA hybrids in  primpol BU‐1A (GAA)10 :Gg RNase H1 (see also   Appendix Fig S5 ) and  primpol BU‐1A (GAA)10 :hPrimPol. An unpaired  t ‐test on three biological replicates was used to compare differences to  primpol BU‐1A (GAA)10  for each matched amplicon. The bar represents the mean, and whiskers represent the SD. *** P  ≤ 0.001, ** P  ≤ 0.01, ns = not significant. Overexpression of chicken RNase H1 prevents (GAA) 10 ‐induced  BU‐1A  epigenetic instability in  primpol  cells. Fluctuation analysis was performed on three  primpol BU‐1A (GAA)10  clones. One‐way ANOVA was used to calculate the significance of differences in  BU‐1  instability between  primpol BU‐1A ΔG4  and other cell lines. **** P  ≤ 0.0001, ns = not significant. Diagram of the RNase H1 hybrid binding domain (HBD)–mCherry fusion and flow cytometry expression profiles of the construct in four clones. Western blots of the same four clones are shown in   Appendix Fig S6 . R‐loop stabilisation induces epigenetic instability of  BU‐1 . Bu‐1a fluctuation analysis of wild‐type cells expressing HBD‐mCherry. The scatter plots pool results from at least two different clones with matched HBD expression. Mean ± SD reported. **** P  ≤ 0.0001, *** P  ≤ 0.001, ns = not significant; one‐way ANOVA.

    Journal: The EMBO Journal

    Article Title: R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

    doi: 10.15252/embj.201899793

    Figure Lengend Snippet: R‐loops promote ( GAA ) 10 ‐dependent epigenetic instability of BU ‐1 DRIP‐qPCR analysis reveals accumulation of R‐loops across the BU‐1 locus in primpol cells. The DRIP signal was calculated as enrichment over RNase H‐treated samples and was normalised to −0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired t ‐test was used to compare differences between matched amplicons in primpol BU‐1A (GAA)10 and the other cell lines indicated. **** P  ≤ 0.0001, ns = not significant. DNA:RNA hybrids in primpol BU‐1A (GAA)10 :Gg RNase H1 (see also Appendix Fig S5 ) and primpol BU‐1A (GAA)10 :hPrimPol. An unpaired t ‐test on three biological replicates was used to compare differences to primpol BU‐1A (GAA)10 for each matched amplicon. The bar represents the mean, and whiskers represent the SD. *** P  ≤ 0.001, ** P  ≤ 0.01, ns = not significant. Overexpression of chicken RNase H1 prevents (GAA) 10 ‐induced BU‐1A epigenetic instability in primpol cells. Fluctuation analysis was performed on three primpol BU‐1A (GAA)10 clones. One‐way ANOVA was used to calculate the significance of differences in BU‐1 instability between primpol BU‐1A ΔG4 and other cell lines. **** P  ≤ 0.0001, ns = not significant. Diagram of the RNase H1 hybrid binding domain (HBD)–mCherry fusion and flow cytometry expression profiles of the construct in four clones. Western blots of the same four clones are shown in Appendix Fig S6 . R‐loop stabilisation induces epigenetic instability of BU‐1 . Bu‐1a fluctuation analysis of wild‐type cells expressing HBD‐mCherry. The scatter plots pool results from at least two different clones with matched HBD expression. Mean ± SD reported. **** P  ≤ 0.0001, *** P  ≤ 0.001, ns = not significant; one‐way ANOVA.

    Article Snippet: The specificity of the pull‐down was tested with RNase H and RNase III treatments prior to immunoprecipitation: one‐third of the digested material was treated with 25 U of RNase H (NEB, M0297), or with 10 U of RNase III (Ambion, AM2290) in appropriate buffers overnight at 37°C, with the subsequent steps performed as described above.

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Over Expression, Binding Assay, Flow Cytometry, Cytometry, Expressing, Construct, Clone Assay, Western Blot

    PrimPol suppresses R‐loop formation in association with  DNA  secondary structure‐forming sequences across the  DT 40 genome Representative normalised DRIP‐seq data in two genes  COL22A1 , spanning over 200 kb, and  MYC . The locations of H‐DNA and G4 motifs are shown below the gene map. Wild type in blue;  primpol  in red. The corresponding RNase H‐treated samples are dashed. See   Materials and Methods  for further details of graphic generation. Metagene analysis of DRIP peak distribution in wild‐type and  primpol  DT40 cells compared with the distribution of the indicated features in the genome. DRIP peak heights in wild type and  primpol  DT40 normalised to  Drosophila  S2 spike‐in.  n  (wild type) = 41,445;  n  ( primpol ) = 48,648. Correlation of normalised DRIP peak heights in the overlapping peaks between wild type and  primpol . Blue line = 1:1 correlation; red line = linear regression through data. Correlation between H‐DNA‐forming sequences and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and  primpol  cells (red). Normalised DRIP peak heights in the genes identified as associating with H‐DNA. Correlation between G4 motifs ([G 3‐5 N 1‐7 ] 4 ) and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and  primpol  cells (red). Normalised DRIP peak heights in the genes identified as associating with G4 motifs ([G 3‐5 N 1‐7 ] 4 ). Data information:  P ‐values calculated with Mann–Whitney  U ‐test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1 st /3 rd  quartile + 1.5*IQR).

    Journal: The EMBO Journal

    Article Title: R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

    doi: 10.15252/embj.201899793

    Figure Lengend Snippet: PrimPol suppresses R‐loop formation in association with DNA secondary structure‐forming sequences across the DT 40 genome Representative normalised DRIP‐seq data in two genes COL22A1 , spanning over 200 kb, and MYC . The locations of H‐DNA and G4 motifs are shown below the gene map. Wild type in blue; primpol in red. The corresponding RNase H‐treated samples are dashed. See Materials and Methods for further details of graphic generation. Metagene analysis of DRIP peak distribution in wild‐type and primpol DT40 cells compared with the distribution of the indicated features in the genome. DRIP peak heights in wild type and primpol DT40 normalised to Drosophila S2 spike‐in. n (wild type) = 41,445; n ( primpol ) = 48,648. Correlation of normalised DRIP peak heights in the overlapping peaks between wild type and primpol . Blue line = 1:1 correlation; red line = linear regression through data. Correlation between H‐DNA‐forming sequences and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Normalised DRIP peak heights in the genes identified as associating with H‐DNA. Correlation between G4 motifs ([G 3‐5 N 1‐7 ] 4 ) and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Normalised DRIP peak heights in the genes identified as associating with G4 motifs ([G 3‐5 N 1‐7 ] 4 ). Data information: P ‐values calculated with Mann–Whitney U ‐test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1 st /3 rd quartile + 1.5*IQR).

    Article Snippet: The specificity of the pull‐down was tested with RNase H and RNase III treatments prior to immunoprecipitation: one‐third of the digested material was treated with 25 U of RNase H (NEB, M0297), or with 10 U of RNase III (Ambion, AM2290) in appropriate buffers overnight at 37°C, with the subsequent steps performed as described above.

    Techniques: MANN-WHITNEY

    PrimPol suppresses R‐loop formation in association with DNA secondary structure‐forming sequences in BOBSC iPS cells Representative normalised RNA DIP‐seq data in the SKI locus. Wild type in blue; primpol in red. The locations of H‐DNA and G4 motifs are shown below the gene map. The corresponding RNase H‐treated samples are dashed. Since so little material was recovered following RNase H treatment, all samples were pooled prior to library generation. Metagene analysis of RNA‐DIP peak distribution in wild‐type and primpol BOBSC cells compared with the distribution of the indicated features in the genome. RNA DIP‐seq peak heights in wild‐type and primpol BOBSC cells normalised to a DT40 spike‐in. n (wild type) = 32,740; n ( primpol ) = 33,721. Correlation of normalised RNA DIP‐seq peak heights in the overlapping peaks between wild type and primpol . Blue line = 1:1 correlation; red line = linear regression through data. Correlation between H‐DNA‐forming sequences and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Correlation between G4 motifs ([G 3‐5 N 1‐7 ] 4 ) and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Normalised RNA DIP‐seq peak heights in the genes identified as associating with H‐DNA. Normalised RNA DIP‐seq peak heights in the genes identified as associating with G4 motifs ([G 3‐5 N 1‐7 ] 4 ). Data information: P ‐values calculated with Mann–Whitney U ‐test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1 st /3 rd quartile + 1.5*IQR).

    Journal: The EMBO Journal

    Article Title: R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

    doi: 10.15252/embj.201899793

    Figure Lengend Snippet: PrimPol suppresses R‐loop formation in association with DNA secondary structure‐forming sequences in BOBSC iPS cells Representative normalised RNA DIP‐seq data in the SKI locus. Wild type in blue; primpol in red. The locations of H‐DNA and G4 motifs are shown below the gene map. The corresponding RNase H‐treated samples are dashed. Since so little material was recovered following RNase H treatment, all samples were pooled prior to library generation. Metagene analysis of RNA‐DIP peak distribution in wild‐type and primpol BOBSC cells compared with the distribution of the indicated features in the genome. RNA DIP‐seq peak heights in wild‐type and primpol BOBSC cells normalised to a DT40 spike‐in. n (wild type) = 32,740; n ( primpol ) = 33,721. Correlation of normalised RNA DIP‐seq peak heights in the overlapping peaks between wild type and primpol . Blue line = 1:1 correlation; red line = linear regression through data. Correlation between H‐DNA‐forming sequences and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Correlation between G4 motifs ([G 3‐5 N 1‐7 ] 4 ) and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Normalised RNA DIP‐seq peak heights in the genes identified as associating with H‐DNA. Normalised RNA DIP‐seq peak heights in the genes identified as associating with G4 motifs ([G 3‐5 N 1‐7 ] 4 ). Data information: P ‐values calculated with Mann–Whitney U ‐test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1 st /3 rd quartile + 1.5*IQR).

    Article Snippet: The specificity of the pull‐down was tested with RNase H and RNase III treatments prior to immunoprecipitation: one‐third of the digested material was treated with 25 U of RNase H (NEB, M0297), or with 10 U of RNase III (Ambion, AM2290) in appropriate buffers overnight at 37°C, with the subsequent steps performed as described above.

    Techniques: DNA Immunoprecipitation Sequencing, MANN-WHITNEY

    Loss of PrimPol leads to unscheduled S phase R‐loop formation Expression of geminin‐tagged chicken RNase H1‐YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 ( X ‐axis). RNase H1‐YFP with or without the geminin degron protein is detected on the Y ‐axis. The RNase H1‐YFP‐geminin degron is degraded in G1. In contrast, RNase H1‐YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replication. Bu‐1a fluctuation analysis of two independently derived primpol BU‐1A (GAA)10 :Gg RNase H1‐YFP‐geminin degron clones. Since the expression of the RNase H1‐YFP‐geminin degron construct is not stable (unlike the RNase H1‐YFP construct without the degron), Bu‐1a expression was assessed separately in the YFP +ve and YFP −ve cells within each clone. Statistical differences calculated the Kruskal–Wallis test. For all panels, at least 36 individual clones were analysed; mean ± SD reported. **** P ≤ 0.0001, *** P ≤ 0.001, ns = not significant. DRIP‐qPCR for R‐loops around the engineered +3.5 (GAA) 10 repeat in BU‐1 in different phases of the cell cycle. The location of the qPCR amplicons is indicated in the map at the top of the panel. The BU‐1 DRIP signal was normalised to −0.5 kb amplicon in G1‐arrested cells ( t = 0 h). See Fig EV4 for representative cell cycle synchronisation profiles. Black: wild type; red: primpol . Error bars = SD. **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. Workflow for the S9.6‐independent detection of newly synthesised R‐loops. See Materials and Methods for details. Validation of analysis of nascent DNA:RNA hybrid formation in BU‐1 locus. Enrichment of 4‐SU‐labelled RNA moiety of DNA:RNA hybrids was calculated relative to input in three independent asynchronous wild‐type (black) or primpol (red) cells, with or without exogenous RNase H treatment. Error bars = SD. ** P ≤ 0.01, * P ≤ 0.05, ns = not significant; unpaired t ‐test. Synchronisation and 4‐SU pulse labelling scheme to identify nascently formed DNA:RNA hybrids. Newly synthesised R‐loops in BU‐1 during S phase in wild type (black) and primpol (red). Error bars represent 1 SD of three biological repeats of the experiment. *** P ≤ 0.001, * P ≤ 0.05; unpaired t ‐test.

    Journal: The EMBO Journal

    Article Title: R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

    doi: 10.15252/embj.201899793

    Figure Lengend Snippet: Loss of PrimPol leads to unscheduled S phase R‐loop formation Expression of geminin‐tagged chicken RNase H1‐YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 ( X ‐axis). RNase H1‐YFP with or without the geminin degron protein is detected on the Y ‐axis. The RNase H1‐YFP‐geminin degron is degraded in G1. In contrast, RNase H1‐YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replication. Bu‐1a fluctuation analysis of two independently derived primpol BU‐1A (GAA)10 :Gg RNase H1‐YFP‐geminin degron clones. Since the expression of the RNase H1‐YFP‐geminin degron construct is not stable (unlike the RNase H1‐YFP construct without the degron), Bu‐1a expression was assessed separately in the YFP +ve and YFP −ve cells within each clone. Statistical differences calculated the Kruskal–Wallis test. For all panels, at least 36 individual clones were analysed; mean ± SD reported. **** P ≤ 0.0001, *** P ≤ 0.001, ns = not significant. DRIP‐qPCR for R‐loops around the engineered +3.5 (GAA) 10 repeat in BU‐1 in different phases of the cell cycle. The location of the qPCR amplicons is indicated in the map at the top of the panel. The BU‐1 DRIP signal was normalised to −0.5 kb amplicon in G1‐arrested cells ( t = 0 h). See Fig EV4 for representative cell cycle synchronisation profiles. Black: wild type; red: primpol . Error bars = SD. **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. Workflow for the S9.6‐independent detection of newly synthesised R‐loops. See Materials and Methods for details. Validation of analysis of nascent DNA:RNA hybrid formation in BU‐1 locus. Enrichment of 4‐SU‐labelled RNA moiety of DNA:RNA hybrids was calculated relative to input in three independent asynchronous wild‐type (black) or primpol (red) cells, with or without exogenous RNase H treatment. Error bars = SD. ** P ≤ 0.01, * P ≤ 0.05, ns = not significant; unpaired t ‐test. Synchronisation and 4‐SU pulse labelling scheme to identify nascently formed DNA:RNA hybrids. Newly synthesised R‐loops in BU‐1 during S phase in wild type (black) and primpol (red). Error bars represent 1 SD of three biological repeats of the experiment. *** P ≤ 0.001, * P ≤ 0.05; unpaired t ‐test.

    Article Snippet: The specificity of the pull‐down was tested with RNase H and RNase III treatments prior to immunoprecipitation: one‐third of the digested material was treated with 25 U of RNase H (NEB, M0297), or with 10 U of RNase III (Ambion, AM2290) in appropriate buffers overnight at 37°C, with the subsequent steps performed as described above.

    Techniques: Expressing, Staining, Derivative Assay, Clone Assay, Construct, Real-time Polymerase Chain Reaction, Amplification

    TFIIS mut Expression Results in an Accumulation of R-Loops (A) RNA or DNA hybrid slot-blot of genomic DNA from TFIIS mut and parental cells, ±RNase H. S9.6 antibody was used to detect RNA or DNA hybrids (upper panel on right) with single-strand DNA antibody (bottom panel) as a loading control. Serial dilutions of genomic DNA (1/1 = 4 μg) were probed with S9.6 antibody for standards (left panel). (B) Fold enrichment in RNA or DNA hybrids compared with control (n = 3). Mean ± SEM (bars) values are shown. p values were determined by unpaired t test. (C and D) DRIP-qPCR analysis of R-loop induction at the SOX4 gene (C) and the SNRPN gene (D) (n = 3). Mean ± SEM (bars) values are shown. p values were determined by two-way ANOVA statistical test. (E) Left: schematic of idealized experiment. Radioactive label is denoted by red dot and the biotin tag on DNA with a black dot. The position of the first adenine in the transcript is also indicated. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and RNase H to yeast TECs assembled in vitro . Ambion RNA size markers are indicated on the left for approximate RNA sizes. Positions of full-length product (FL), R-loops, and cleavage products are indicated on the right. (F) Left: experimental scheme; similar to that of (E) but involving purification via the biotin tag after RNase H digestion. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and RNase H to yeast TECs assembled in vitro . Approximate RNA sizes RNA and position of R-loops are indicated on the right and next to relevant lanes. Asterisk-bar denotes irrelevant pausing sites of unknown origin, including IC1 and IC2. See Figure S5 for detailed schematic explanations.

    Journal: Molecular Cell

    Article Title: Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability

    doi: 10.1016/j.molcel.2019.07.037

    Figure Lengend Snippet: TFIIS mut Expression Results in an Accumulation of R-Loops (A) RNA or DNA hybrid slot-blot of genomic DNA from TFIIS mut and parental cells, ±RNase H. S9.6 antibody was used to detect RNA or DNA hybrids (upper panel on right) with single-strand DNA antibody (bottom panel) as a loading control. Serial dilutions of genomic DNA (1/1 = 4 μg) were probed with S9.6 antibody for standards (left panel). (B) Fold enrichment in RNA or DNA hybrids compared with control (n = 3). Mean ± SEM (bars) values are shown. p values were determined by unpaired t test. (C and D) DRIP-qPCR analysis of R-loop induction at the SOX4 gene (C) and the SNRPN gene (D) (n = 3). Mean ± SEM (bars) values are shown. p values were determined by two-way ANOVA statistical test. (E) Left: schematic of idealized experiment. Radioactive label is denoted by red dot and the biotin tag on DNA with a black dot. The position of the first adenine in the transcript is also indicated. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and RNase H to yeast TECs assembled in vitro . Ambion RNA size markers are indicated on the left for approximate RNA sizes. Positions of full-length product (FL), R-loops, and cleavage products are indicated on the right. (F) Left: experimental scheme; similar to that of (E) but involving purification via the biotin tag after RNase H digestion. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and RNase H to yeast TECs assembled in vitro . Approximate RNA sizes RNA and position of R-loops are indicated on the right and next to relevant lanes. Asterisk-bar denotes irrelevant pausing sites of unknown origin, including IC1 and IC2. See Figure S5 for detailed schematic explanations.

    Article Snippet: WT-TFIIS and TFIISmut were added for 3 min, followed by addition of 5U of RNase H (NEB, M0297S) and incubated at RT for 30 min. For the experiment shown in E, the TEC/DNA scaffolds were purified via the biotin-tag on the non-transcribed strand after RNase H treatment to ensure that only anterior R-loops were detected (the label on posterior R-loops is released into the supernatant fraction due to RNase H cleavage).

    Techniques: Expressing, Dot Blot, Real-time Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, In Vitro, Purification