The TN-RCA reaction product was digested with MseI (NEB) (10 U) for 20 min at 37 °C in the presence or absence of cutting primer, Msecutprimer: 5’-TTTATCTTAA CTCACCAACT-3’ (underlined: MseI recognition site), and the enzyme subsequently inactivated at 65 °C for 20 min. Lambda exonuclease (NEB) (0.5 U), exonuclease III (NEB) (20 U), exonuclease VIII (NEB) (2 U), T7 exonuclease (2 U), RNase H (NEB) (1 U), RNase A (Thermo Scientific, Waltham, MA, USA) (10 U), RNase A/T1 Mix (Thermo Scientific) (1 U), RNase T1 (Invitrogen/Ambion) (1 U), ShortCut RNase III (NEB) (0.4 U) were added to the TN-RCA reaction mixture after the ligation step was completed, or at various times during the TN-RCA reaction. In some experiments, ShortCut RNase III (NEB) (0.4 U) was also added before the ligation step to fragment the target genomic Zika RNA. Random hexamers (Exo-Resistant Random Primer; 0.2 μL of 500 μM stock; Thermo Scientific) also was added after the ligation step.
Shortcut rnase 3
Manufactured by New England Biolabs
Sourced in United States
ShortCut RNase III is a recombinant ribonuclease III enzyme from Escherichia coli that cleaves double-stranded RNA (dsRNA) into shorter fragments.
Automatically generated - may contain errors
Lab products found in correlation
Rnase h, by New England Biolabs (5 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by New England Biolabs (3 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Megascript rnai kit instructions, by Thermo Fisher Scientific (2 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rnase a, by New England Biolabs (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Fluoroshield mounting medium with dapi, by Abcam (1 mentions)
Rnase t1, by Thermo Fisher Scientific (1 mentions)
Fluoview fv1200 confocal microscope, by Olympus (1 mentions)
T7 rnap, by New England Biolabs (1 mentions)
Protoscript 2 first strand cdna synthesis kit, by New England Biolabs (1 mentions)
Recombinant human il 2, by Merck Group (1 mentions)
Slurry protein a, by GE Healthcare (1 mentions)
Rnase 3, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
33 protocols using shortcut rnase 3
1
Optimization of TN-RCA RNA Detection
2
DRIP-seq Protocol for Detecting RNA-DNA Hybrids
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Genomic DNA from HCT116 and HCT116-TOP3B-KO cells treated with DMSO or NSC690634 (100 µM, 4 h) was extracted using the DNA–RNA immunoprecipitation sequencing (DRIP) protocol as described previously (29 (link)). Briefly, cells were lysed in TE buffer containing SDS and proteinase K (at 37 °C overnight) before being extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and ethanol precipitated. Genomic DNA was resuspended in TE buffer and digested using a cocktail of restriction enzymes (HindIII, SspI, EcoRI, BsrGI, and Xbal; 30 U each), treated with RNase A (10 µg/mL) and shortcut RNase III (2 units, New England Biolabs) before purification again with phenol/chloroform/isoamyl alcohol (25:24:1). For control samples, 10 µg of genomic DNA was treated with 20 U of RNase H at 37 °C for 3 h. The resulting genomic DNA samples were spotted on nitrocellulose 0.45-μm membranes (Bio-Rad Laboratories, CAT#: 1620115), cross-linked and blocked with PBS-Tween (0.1%) buffer containing 5% nonfat milk. The membrane was probed with mouse S9.6 antibody (1:500 dilution, at 4 °C overnight) and developed using standard enhanced chemiluminescence techniques. The same samples probed with anti-dsDNA antibodies served as loading controls.
3
Double-stranded RNA Digestion with ShortCut RNase III
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Double-stranded RNAs were digested with ShortCut RNase III (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol.
4
Preparation of Diced siRNA Pools
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The dsRNA was digested with Short Cut RNase III (New England Biolabs, United States) to prepare diced-siRNA pools according to the manufacturer’s instruction. Briefly, 10 μg dsRNA were digested with Short Cut RNase III in a 100 μL reaction system (dH2O, 10X ShortCut Reaction Buffer: 10 μL, dsRNA: 10 μg, ShortCut RNase III: 10 μL, 10X MnCl2: 10 μL) at 37°C for 20 min. Then 10 μL 10X EDTA was added to stop the reaction. The diced small RNAs were purified by precipitation using nuclease-free glycogen, one-tenth volume of 3M sodium acetate (pH 5.2) and 3 volumes of cold ethanol. Diced siRNA were stored at -80°C (Kalleda et al., 2013 (link)).
5
Immunofluorescence Imaging of DNA:RNA Hybrids
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Cells were plated on glass cover slips. Cover slips were washed 3 times in cold PBS and fixed in cold 4% paraformaldehyde (Alfa Aesar) in PBS for 10 min. Cover slips were then washed 3 times in cold PBS and permeabilised with cold 0.2% Triton X-100 in PBS (PBST) for 10 min. Slides were then washed 3 times in cold PBS and blocked in 10% FBS in PBS for 2 h at 4°C. Primary antibodies were diluted to the appropriate concentration in 10% FBS in PBS and incubated overnight at 4°C in a humidified chamber. Cover slips were then washed 3 times in PBST. Coverslips were incubated for 2 h at RT with secondary antibodies diluted in 0.15% FBS in PBS. Coverslips were then washed 3 times in PBST in the dark. Coverslips were then washed a final time in PBS before mounting on slides with Fluoroshield Mounting Medium with DAPI (Abcam). Slides were imaged using an Olympus Fluoview FV1200 confocal microscope with a 60X objective lens.
For S9.6 Immunofluorescence experiments, after fixation permeabilisation with 0.2% Triton X-100, cells were treated with RNase pre-treatment solution (0.1% BSA, 3mM MgCl2, RNAse T1 (Thermo) 1:200 (v/v), ShortCut RNAseIII (NEB) 1:200 (v/v) in PBS) for 2 h at RT (Alagia et al., 2022 ). Subsequent blocking and antibody incubation steps were then carried out as above.
For S9.6 Immunofluorescence experiments, after fixation permeabilisation with 0.2% Triton X-100, cells were treated with RNase pre-treatment solution (0.1% BSA, 3mM MgCl2, RNAse T1 (Thermo) 1:200 (v/v), ShortCut RNAseIII (NEB) 1:200 (v/v) in PBS) for 2 h at RT (Alagia et al., 2022 ). Subsequent blocking and antibody incubation steps were then carried out as above.
6
Dg vATPase A siRNA Synthesis Methods
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Dg vATPase A siRNAs were synthesized by either in vitro digestion of long dsRNAs (method 1) or by chemical synthesis of 27-mer blunt dsRNAs (method 2). In method 1, long dsRNAs for R1 and R2 of the Dg vATPase A gene and lacZ control gene (120 μg of each dsRNA) were incubated with 0.2 units/μl ShortCut® RNase III (New England BioLabs, Ipswich, MA, USA) for 3 h at 37°C to produce a heterogeneous mix of short (18–25 bp) siRNAs. Reactions were stopped with EDTA according to the manufacturer’s protocol and precipitated in ethanol, following which the size distribution of the digested RNAs was validated by electrophoresis using a 4% agarose gel. In method 2, Dicer-substrate siRNAs (27-mer blunt dsRNAs) were designed based on the coding sequence of the Dg vATPase A gene and lacZ control gene using the Eurofins Genomics siMAX siRNA design tool, commercially synthesized and then annealed by Eurofins Genomics (Eurofins Genomics, Ebersberg, Germany). The sequence of each siRNA is shown in Additional file 4 : Figure S2.
7
Generating Endoribonuclease-Digested siRNAs
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Endoribonuclease-digested short interfering RNAs (esiRNAs) were generated as previously described [68 (link)–70 (link)]. Briefly, DNA sequences were identified within the target genes and screened for unique nucleotide sequence via DEQOR [71 ]. siRNAs were in vitro transcribed using T7 polymerase using cDNA collected from wildtype murine ES cells as a template. siRNAs were then digested with ShortCut RNase III (NEB) into esiRNAs, which were then purified using a PureLink RNA Mini Kit (Invitrogen). Transient transfections were performed on 10 cm plates using 25 µL of Lipofectamine 3000 (ThermoFisher) and 3.5 µg of esiRNAs for 48 h and validated by RT-qPCR.
8
Endoribonuclease-Prepared siRNA Transfection
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Endoribonuclease-prepared siRNAs were prepared as described (60 (link)). Briefly, cDNA from mESCs was used as a template to amplify cDNA that targets each gene with T7 anchor sequence added. In vitro transcription was then performed using T7 RNAP (New England Biolabs; M0251L). RNA was digested by ShortCut RNase III (New England Biolabs; M0245L) to generate a pool of small siRNAs, which was purified using a PureLink RNA Mini Kit (Thermo Fisher Scientific; 12183020). For transfection, 400 ng of endoribonuclease-prepared siRNA was mixed with 0.4 ml of serum-free medium and 4 μl of Lipofectamine 2000 (Thermo Fisher Scientific; 11668-019), and after 15 min of incubation, 2.8 × 105 mESCs were added and the mixture was plated in one well of a gelatinized 6-well plate. Media were replaced ∼16 h later, and cells were harvested 48 h after transfection.
9
Nuclear RNA Extraction and cDNA Synthesis
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The nuclear fraction from HepG2 cells was suspended in 200 µl of RIPA buffer (150 mM NaCl, 1% NP40, 0.1% SDS, 20 mM MnCl2, 50 mM Tris-Cl at pH 8, 5 mM EDTA at pH 8) and then frozen and thawed three times. After centrifugation, 10 units of Shortcut RNAse III (NEB) which specifically digests double-stranded RNA were added and incubated at 37 °C for 30 minutes. RNAs were then isolated using the ReliaPrep miRNA Cell and Tissue Miniprep System according to the manufacturer’s protocol. cDNA synthesis was carried out using ProtoScript II First Strand cDNA Synthesis Kit (NEB) and Oligo d(T)/ random primers mix (NEB).
10
In vitro dsRNA Digestion by RNase III
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