DsRNA for Illumina sequencing was extracted from 20 g ground fungal material, stored at − 80 °C, based on a modified protocol of Morris and Dodds [23 (link)] as described by Lesker et al. [24 (link)], apart from using a different cellulose (acid-washed powder for column chromatography [Merck; Darmstadt, Germany; product nr. 22,184]). 20 mL eluate was digested first with 20 U Rnase T1 (Roche; Basel, Switzerland) and then with 40 U DNAse I (Roche; Basel, Switzerland) at 37 °C for 30 min each. DsRNA extracts were centrifuged and suspended in 25 µL Tris (5 mM). Subsequently, 20 µL extract was checked with 5 µL of GelRed® (Biotium; Fremont, CA, USA) dye in 1.5% agarose gel electrophoreses. For virus detection by RT-PCR and RNA end determination, a simpler protocol for whole nucleic acid extraction was used, following the protocol of Menzel et al. [25 (link)].
Rnase t1
Manufactured by Roche
Sourced in Switzerland, United States
RNase T1 is an endoribonuclease that cleaves single-stranded RNA at guanine residues. It specifically hydrolyzes the phosphodiester bonds on the 3' side of guanine residues in RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Rnase t2, by Thermo Fisher Scientific (2 mentions)
Lithium chloride (licl), by Thermo Fisher Scientific (2 mentions)
Synthetic dna oligonucleotides, by Merck Group (2 mentions)
γ 32p atp, by PerkinElmer (2 mentions)
α 32p utp, by PerkinElmer (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Trizol, by Merck Group (2 mentions)
Thermomixer, by Eppendorf (2 mentions)
Rnase a, by Roche (2 mentions)
Gelred, by Biotium (1 mentions)
Dnase 1, by Roche (1 mentions)
Ultrapure distilled water, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 c5 maleimide, by Thermo Fisher Scientific (1 mentions)
Inactivation precipitation buffer, by Thermo Fisher Scientific (1 mentions)
Heparin, by Merck Group (1 mentions)
His tag protein purification kit, by Merck Group (1 mentions)
Escherichia coli trna, by Roche (1 mentions)
Riboflavin, by Thermo Fisher Scientific (1 mentions)
E coli trnatyr, by Merck Group (1 mentions)
20 protocols using rnase t1
1
Purification and Characterization of Fungal dsRNA
2
Adenovirus Genome Extraction Protocol
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A 400 μl volume of CsCl density gradient purified adenovirus was transferred into a 1.5-ml tube and treated for at least 3 h at 37 °C with 200 ug/ml of proteinase K (Invitrogen, Cat#: AM2546), 0.5% SDS and 500–1,000 units of RNase T1 (Roche, Cat# 109193). The mixture was then treated with a PCIA extraction solution (phenol: chloroform: isoamyl alcohol = 25: 24: 1) and the released adenovirus genome was subsequently precipitated with ethanol and then resuspended in DNAase-free UltraPure Distilled Water (Invitrogen, Cat#: 10977-015).
3
RNA Footprinting Analysis of CsrA Binding
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RNA was synthesized using a MEGAscript kit and a PCR fragment containing a T7 promoter derived from primer HY3. The template was a DNA fragment containing csrR sequence extending from 32 to +84 relative to the start of transcription. Binding reactions (10 µl) containing 5 nM csrR RNA and the indicated concentrations of CsrA-H6 were otherwise identical to those in the gel shift assay. After the initial binding reaction, 0.075 U RNase T1 (Roche) was added to the reaction mixtures, and incubation continued for 15 min at 37°C. Reactions were terminated by addition of 10 µl of gel loading buffer, and reaction mixtures were placed on ice. Partial alkaline hydrolysis and RNase T1 digestion ladders of each transcript were prepared as described previously (59 (link)). After samples were fractionated through the use of standard 6% polyacrylamide sequencing gels, radioactive bands were visualized with a phosphorimager.
4
Probing RNA Structure with Ribonuclease Digestion
5
RNase T1 Footprinting of CrPV11 RNA
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40,000 cpm of 5’ end-radiolabeled CrPV11 (domain III only) WT, G-rich, and △3 RNAs were folded by heat-cooling in 30 mM HEPES-KOH pH 7.5, 10 mM MgCl2, in the presence of 1 μg carrier tRNA. RNase T1 (Roche) digestion was performed by adding 0.1 U of enzyme and incubating at 37°C for 2 min. RNAs were ethanol precipitated overnight and resuspended to equal counts per microliter in 1X TBE + 9M urea loading buffer. RNase T1 (G) (denaturing) ladders for each RNA and a hydrolysis ladder of the WT CrPV11 RNA were generated as previously described (Kieft et al., 1999 (link)). Samples were loaded on a 12% polyacrylamide-urea sequencing gel (0.4 mm gel thickness) and run for 2 hr at 65 W. For analysis, data were normalized to total amount of radiation in each lane before subtracting the appropriate non-native T1 cleavage signal (G ladders) from the native T1 cleavage signal.
6
RNA Oligonucleotide Labeling and Purification
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RNA oligonucleotides were labelled with Alexa Fluor 546 C5-maleimide (Thermo Fisher Scientific, Waltham, MA, USA) using the 5′ EndTag Nucleic Acid Labelling System (Vector Laboratories, Burlingame, CA, USA). Labelled RNAs were purified using a denaturing polyacrylamide gel and precipitated with ethanol in the presence of 20 µg glycogen. Five pmol labelled RNAs in a transcription-buffer (T-buffer) containing 50 mM HEPES-KOH (pH 7.6), 5 mM magnesium acetate, 100 mM potassium glutamate, 2 mM spermidine and 0.01% (v/v) Tween 20 were incubated at 70 °C for 5 min and cooled to 37 °C at a rate of 1 °C min−1. The samples were incubated with 0.02 U of RNase T1 (Roche, Basel, Switzerland) at 37 °C for 10 min and electrophoresed on a 20% denaturing polyacrylamide gel at 70 °C. The fluorescence signal in the gel was imaged using a FLA-5100 fluorescence image scanner (Fuji Film, Tokyo, Japan) with 532 nm excitation and 575 nm emission.
7
RNA-Protein Binding Assays with S6:S18 and S18
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For S6:S18 nuclease protection assays, the RNA–protein binding reactions identical to those used for EMSA assays above (50 μL reaction volume with S6:S18 dimer concentrations of 0, 50, 100, 300, and 600 nM) were assembled. For S18 nuclease protection assays binding reactions with S18 concentrations of 0, 25, 50, 100, 300, and 600 nM were similarly assembled. After protein-RNA incubation, each reaction was treated with 1 μL RNase A and V1 (purchased from Ambion: 20 pg and 0.002 units, respectively) for 10 min and 5 min, respectively, at 25°C. The nuclease was inactivated with inactivation/precipitation buffer (Ambion), and RNA fragments were recovered by ethanol precipitation. Precipitated RNAs were resuspended in 10 μL water and 10 μL urea loading solution (10 M urea, 1.5 mM EDTA). Ten microliters of each reaction was loaded on a 12% denaturing Acrylamide/Bis gel and run at 35W at 25°C. Partial alkaline hydrolysis reactions were generated by incubating RNA in 50 mM Na2CO3 pH 9.0, 1 mM EDTA for 10 min at 95°C. Denaturing T1 reaction was conducted by incubating 1U of RNAse T1 (Roche) in 25 mM Na Citrate pH 5.0, 5.5 M urea for 15 min at 55°C. The gel was dried and examined using a GE Healthcare STORM 820 phosphorimager and ImageQuant.
8
Iron Regulatory Protein Binding Assay
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RNA-EMSAs were performed by incubating whole-cell lysates (15 µg) with a [32P]GTP-labeled ferritin L-IRE probe in 10 mM HEPES, pH 7.6, 3 mM MgCl2, 40 mM KCl, 5% glycerol, 1 mM DTT for 20 min at room temperature66 (link). Heparin (Sigma) (50 µg/µl) and RNase T1 (Roche) (1 U/µl) were added simultaneously to the lysates for 10 min. Irp1- and Irp2-RNA complexes were resolved by 5% non-denaturing PAGE (acrylamide:methylene bisacrylamide ratio, 60:1), and the dried gel exposed to a PhosphorImager screen for analysis.
9
Purification of Ribonuclease Cusativin
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Escherichia coli tRNA and RNase T1 were procured from Roche Diagnostics (Indianapolis, IN, USA). E. coli tRNATyr was obtained from Sigma-Aldrich (St. Louis, MO, USA). Riboflavin and all other chemicals were acquired from Fisher Scientific (Fairlawn, NJ, USA) unless otherwise specified. Ribonuclease cusativin was purified from the overexpression strain of E. coli on a nickel column using a His-tag protein purification kit from EMD Millipore (Burlington, MA, USA) as described [51 (link)].
10
Radiolabeled Nucleotides for Biochemical Assays
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Lithium chloride was purchased from Acros Organics. Ammonium chloride was purchased from Macron Fine Chemicals. Calcium chloride dihydrate and magnesium chloride hexahydrate were purchased from J.T. Baker. All salts were 99% pure or greater, except calcium chloride dihydrate was 97% or greater. [γ-32P]ATP and [α-32P]UTP were purchased from PerkinElmer. All other chemicals and synthetic DNA oligonucleotides were purchased from Sigma-Aldrich. RNase T1 was purchased from Roche. All other enzymes were purchased from New England Biolabs unless otherwise indicated.
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