Two hundred fifty nanograms of DNA containing R-loops from each strain was incubated with 1 unit of RNase H (#M0297, NEB) or mock for no RNase H digestion control, in 1× RNase H digestion buffer for 12 h at 37°C. DNA was phenol extracted and ethanol precipitated and reconstituted in 10 µL TE buffer. Five microliters 1× or 0.2× titration of DNA solution was loaded to nitrocellulose membrane, air dried for 30 min, and baked for 2 h at 80°C. The membrane was blocked in 5% BSA for 1 h and incubated with 1:1000 S9.6 antibody (gift from the Proudfoot laboratory) (Boguslawski et al. 1986 (link)) overnight at 4°C in order to detect RNA–DNA hybrids.
Rnase h
Manufactured by New England Biolabs
Sourced in United States, United Kingdom, Germany
RNase H is a DNA-RNA hybrid-cleaving enzyme. It specifically hydrolyzes the phosphodiester bonds in the RNA strand of an RNA-DNA hybrid, thereby separating the two strands.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3, by Thermo Fisher Scientific (19 mentions)
Dnase 1, by New England Biolabs (17 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (14 mentions)
Rnase a, by Thermo Fisher Scientific (13 mentions)
Trizol, by Thermo Fisher Scientific (13 mentions)
Dna polymerase 1, by New England Biolabs (12 mentions)
Turbo dnase, by Thermo Fisher Scientific (12 mentions)
Rnase h buffer, by New England Biolabs (11 mentions)
Dnase 1, by Thermo Fisher Scientific (11 mentions)
E coli dna polymerase 1, by New England Biolabs (10 mentions)
Dutp solution, by Thermo Fisher Scientific (10 mentions)
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Dntps, by Thermo Fisher Scientific (9 mentions)
Rnase a, by Merck Group (7 mentions)
Rnase 3, by New England Biolabs (7 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (7 mentions)
Rnasin, by Promega (6 mentions)
T4 rna ligase, by New England Biolabs (6 mentions)
Rnase a, by New England Biolabs (6 mentions)
Rnase if, by New England Biolabs (6 mentions)
344 protocols using rnase h
1
Quantitative RNA–DNA Hybrid Analysis
2
In vitro trans-splicing Reaction Assay
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In vitro trans-splicing reactions were run for 60 min (as described above) and quenched with EDTA to a final concentration 1.2-fold higher than MgCl2. Eight microliters of each reaction were added to 2 μL of the indicated DNA oligonucleotide, to a final concentration of 1 μM DNA. The oligonucleotide was allowed to anneal to the products of the splicing reaction by heating to 80°C for 2 min, followed by cooling to 25°C at 0.1°C/sec. Two μL MgCl2 were added to the reaction in stoichiometric equivalent to the EDTA present. Fifty microliters of RNase H reactions contained the 12 μL annealing reaction, 1× RNase H Reaction Buffer and 2.5 units RNase H (New England Biolabs) and were incubated at 37°C for 1 h. Reactions were purified by ethanol precipitation and separated by denaturing 6% PAGE. Gels were imaged on a phosphoimager (Bio-Rad). DNA oligonucleotides were designed to bind to the following nucleotide positions along the CAT pre-mRNA, where position 1 is the A of the translation start codon: 35–68, 81–108, 112–139, 117–144, 122–149, 127–154, 132–162, 147–177, 153–182, 158–190, 162–194, 191–221, 245–276, 279–307, 290–316, 315–341, 321–350, 330–359, 341–370, 350–378, 360–391, 370–401, 380–411, 385–416, 390–421, 487–516, 624–653, and 739–770.
3
RNA-FISH Probe Amplification Protocols
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(Figure 1—figure supplement 5 ). After probe hybridization and washing with PBS, samples were treated in 50 μl final volume for either RNAseIII treatment: 1X RNAseIII buffer, 1.5 μl Shortcut RNAseIII (New England Biolabs, cat. M0245s), and 1X MnCL2 or RNAseH treatment: (1X RNAseH buffer, 1.5 μl RNAseH (New England Biolabs, cat. M0297S) at 37°C for 2 hr. Samples were then blocked with 2x PBT:WBR 1 hr then processed as per protocol ‘TSA amplification for RNA-FISH probe detection’ (protocol three above). Three embryos treated with RNAseH were imaged, while six treated with RNAseIII were imaged.
4
RNA Hydrolysis and Purification
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5
Quantification of DNA-RNA Complexes
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DNA-RNA complexes were first generated by adding 20,000 cpm of 32P-labelled MITF 1550–1740 RNA probe to separate tubes containing 2 μg of DNA oligonucleotides (MHO-1 or MHO-7) or 2 μg yeast tRNA as control. The nucleic acid samples were precipitated and resuspended in 10 μl of Hybridization Buffer III (Ambion RPA III kit) followed by denaturation at 95°C for 3 min, and hybridization overnight at 42°C. Three 150 μl RNase H solutions containing 7.5 U of RNase H (New England Biolabs) in 1 X RNase H buffer were prepared and mixed with each hybridization reaction and incubated at 37°C for 1 hour, followed by heat inactivation at 65°C for 20 min. Digested RNA was then precipitated at -20°C for 1 hour after adding 225 μl of RNase Inactivation/precipitation III solution (Ambion RPA III kit), followed by 30 min centrifugation at 10,000 x g. RNA pellets were dried and suspended in 10 μl of Gel Loading Buffer II (RPA III kit) and fully denatured by heating at 95°C for 3 min. RNA samples were then resolved by gel electrophoresis on an 8% denaturing (7 M urea) polyacrylamide gel and exposed overnight to phosphor screen before visualization using the Cyclone Plus phosphorimager system (PerkinElmer).
6
RNase H Digestion and Analysis
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To perform RNase H digestion, we followed a protocol that allows comparison of RNase H treated and untreated NA at the same temperature (7 (link)). 10 μg of chromatin-associated, phenol/chloroform-isolated NA were incubated for 2 h at 37°C with 13 U of RNase H (NEB) in 1× buffer (NEB) in a total volume of 30 μl. The untreated (i.e. without RNase H) control (10 μg) was also incubated for 2 h at 37°C in 1× NEB buffer. RNase H treated and untreated samples were then double DNase I digested and processed for directed RT-qPCR.
7
Purification and RNase H Analysis of mRNA
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Approximately 20 µg of total RNA was used as starting material for each reaction. Total RNA was isolated with TRIzol, followed by extraction with acid phenol/chloroform and chloroform. RNA was precipitated, washed in 70% ethanol, resuspended, and then treated with RQ1 DNase (Promega), purified with an RNeasy kit (Qiagen), and mRNA was isolated using Oligotex (Qiagen) and precipitated. For RNase H assays, RNA was resuspended in annealing buffer (1 mM EDTA [pH 8] [Ambion], 0.2 M KCl) with and without 100 pmol of oligo(dT21), heated to 95°C for 3 min, and allowed to anneal at room temperature for 20 min. RNaseH buffer and RNase H (New England Biolabs) were then added, and reactions were incubated at 37°C for 45 min. Reactions were then adjusted to 0.5% SDS, 10 mM EDTA, and 250 µg/mL proteinase K (New England Biolabs), and were incubated at 37°C for 30 min. RNA was purified by acid phenol chloroform and chloroform extraction and analyzed by 1.2% agarose–formaldehyde gel electrophoresis and Northern blot analysis.
8
RNA/DNA Hybrid Slot Blot Analysis
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RNA/DNA hybrid slot blot was performed as described (Cristini et al., 2018 (link); Kotsantis et al., 2016 (link)). RNase H sensitivity was carried out by incubation with 1.7 U of RNase H (NEB, M0297) per µg of genomic DNA for 2.5 h at 37°C. For loading control, 250 ng of genomic DNA was heated at 95°C for 10 min and loaded on the Slot Blot, and the membrane was denatured in 0.5 M NaOH and 1.5 M NaCl for 5 min and neutralized for 2 min in 0.5 M Tris-HCl, pH 7.2, and 1.5 M NaCl. The membrane was probed with αssDNA (MAB3034; Millipore) after UV crosslinking and saturating. Images were acquired with LAS-4000 (Fujifilm) or by chemiluminescence using autoradiography. S9.6 and ssDNA signals were quantified using Image Studio Lite software (Li-COR Biosciences).
9
Targeted RNA Digestion Assay
10
Removing Poly(A) Tails from mRNA
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To remove poly(A) tails from the 3′end of mRNAs, 10 μg total RNA was mixed with 3 μg oligodT18. RNA:DNA duplexes were generated in a thermocycler according to the following program: 3 min at 70 °C, 5 min at 42 °C, ramp down to 25 °C with 0.1 °C/s. RNase H digestion was performed using 5 U RNase H (NEB) for 20 min at 37 °C in the presence of 40 U RNasin (Promega). RNA was recovered by phase separation using phenol:chloroform:isamylalcohol (25:24:1; AppliChem) and precipitated following addition of 1 μl GlycoBlue coprecipitant (Thermo Fisher Scientific), 0.1 volumes 5M NaOAc, and 2.5 volumes 100% EtOH. RNA was precipitated O/N at −20 °C and further processed as described above.
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