Rnaseout

Manufactured by Thermo Fisher Scientific

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RNaseOUT is a recombinant ribonuclease inhibitor protein that protects RNA from degradation by RNase enzymes. It is a highly effective inhibitor of RNase A, RNase B, and RNase C.

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963 protocols using rnaseout

Subcellular Fractionation and RNA Isolation

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Approximately 2.5 × 10⁶ cells were pelleted, washed with PBS, resuspended in 250 μl Lysis Buffer (15 mM HEPES pH7.5, 10 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, 0.5 mM EGTA, 250 mM Sucrose, 0.4% Igepal, 1 mM DTT, 40 U/ml RNaseOUT (Invitrogen), protease inhibitor cocktail [Roche]) and incubated on ice for 20 min. Nuclei were centrifuged at 2,000 g for 10 min at 4°C and the supernatant was collected as the cytoplasmic fraction. Nuclei were then resuspended in 50 μl Nuclei Lysis Buffer (10 mM HEPES pH7.5, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 40 U/ml RNaseOUT (Invitrogen), protease inhibitor cocktail [Roche]) and incubated on ice for 5 min. Nuclei were pelleted at 17,000 g for 5 min at 4°C and the supernatant was removed as the nucleoplasm fraction. The pellet was then resuspended in 50 μl Salt Extraction Buffer (25 mM HEPES pH7.5, 10% glycerol, 420 mM NaCL, 5 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, 40 U/ml RNaseOUT (Invitrogen), protease inhibitor cocktail [Roche]) and incubated for 30 min at 4°C with rotation. The sample was then centrifuged at 17,000 g for 20 min at 4°C. The supernatant was collected as the salt extracted fraction and the pellet resuspended in 50 μl Salt Extraction Buffer to generate the chromatin fraction. RNA was isolated from each fraction using the Qiagen Mini RNeasy kit following the manufacturer's instructions.

Vance K.W., Sansom S.N., Lee S., Chalei V., Kong L., Cooper S.E., Oliver P.L, & Ponting C.P. (2014). The long non-coding RNA Paupar regulates the expression of both local and distal genes. The EMBO Journal, 33(4), 296-311.

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Proteomic Profiling of LncHrt Interactome

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To identify the binding partners of LncHrt, we used a tagged-RNA pulldown assay, as described [37 (link), 64 (link)]. Sense and antisense strands of streptavidin-binding S1m DNA were synthesized (Tsingke), annealed, and cloned into pCDH-MSCV at a cloning site, which was then used to insert sequences encoding LncHrt. The constructs expressing S1m, S1m-LncHrt as well as those expressing untagged LncHrt and EGFP were packaged into lentivirus using PEI (1:4) and then infected the cardiac fibroblasts with 20 MOI for 48 h. Cells were then harvested in SA-RNP lysis buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 2 mM DTT, 50 U/mL RNase OUT (Life Technologies) 50 U/mL Superase IN (Ambion) and 1 × complete protease inhibitor tablet (Roche). Streptavidin–Sepharose beads were blocked with 500 ng/µL yeast tRNA and 1 mg/mL BSA in SA-RNP lysis buffer before being added into cell lysates and being incubated at 37 °C for 2 h on a rotator. The beads were then pelleted and washed five times with SA-RNP washing buffer (20 mM Tris–HCl (pH 7.5), 300 mM NaCl, 5 mM MgCl2, 2 mM DTT, 50 U/mL RNase OUT (Life Technologies, NY, USA), 50 U/mL Superase IN (Ambion) and 1 × complete protease inhibitor tablet (Roche). After the last wash, RNA-bound proteins were then boiled in 50 µL 3 × LDS sample buffer (Life Technologies) and used for mass spectrometry.

Liu N., Kataoka M., Wang Y., Pu L., Dong X., Fu X., Zhang F., Gao F., Liang T., Pei J., Xiao C., Qiu Q., Hong T., Chen Q., Zhao J., Zhu L., He J., Hu X., Nie Y., Zhu W., Yu H., Cowan D.B., Hu X., Wang J., Wang D.Z, & Chen J. (2021). LncRNA LncHrt preserves cardiac metabolic homeostasis and heart function by modulating the LKB1-AMPK signaling pathway. Basic Research in Cardiology, 116(1), 48.

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Intracellular Sorting and RNA Extraction

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Intracellular sorting followed by RNA extraction was adapted from a previously described protocol ^{30 (link)}. NHBE cells in ALI culture (day 21) were trypsinized and fixed with 4% paraformaldehyde in PBS with RNASEout (1:100, Life Technologies) for 30 minutes at room temperature. Following 2 washes with wash buffer (PBS supplemented with 0.5% BSA, 0.05% Tween-20 and 1:100 RNASEout), cells were incubated with either anti-β-tubulin IV (Sigma) or anti-MUC5AC (Thermo Scientific) antibodies for 1 hour at 4°C in PBS with 1% BSA, 0.05% Tween-20 and RNASEout (1:25). Following 3 washes with wash buffer, cells were incubated with secondary antibody (1:100), washed again for another 3 washes and sorted by FACS (BD FACS-Aria Sorter). RNA was then extracted using the PFFE Kit (Qiagen) and transcribed using random hexamers in the First-strand Synthesis Kit (Life Technologies). Quantitative PCR was performed for GSDMB using primers above and with the following additional primers (FOXJ1 and MUC5AC) to determine whether efficient separation of ciliated or goblet cells was achieved.

Panganiban R.A., Sun M., Dahlin A., Park H.R., Kan M., Himes B.E., Mitchel J.A., Iribarren C., Jorgenson E., Randell S.H., Israel E., Tantisira K., Shore S., Park J.A., Weiss S.T., Wu A.C, & Lu Q. (2018). A functional splicing variant associated with decreased asthma risk abolishes the ability of gasdermin B (GSMDB) to induce epithelial cell pyroptosis. The Journal of allergy and clinical immunology, 142(5), 1469-1478.e2.

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Circular RT-PCR for Poly(A) Analysis

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The circular RT-PCR (cRT-PCR) protocol for the poly(A) analyses was adapted from Rosenthal et al. (2018) (link). Five micrograms of total RNA had the 5′ cap removed using RppH (New England Biolabs, Ipswich, USA) in a reaction containing NEB Thermopol buffer (New England Biolabs, Ipswich, USA) and RNAseOut (Thermo Fisher Scientific, Waltham, USA) for 1 h at 37°C. To stop the de-capping reaction, RQ1 DNAse stop solution (Promega, Madison, USA) was added and samples were heated for 5 min at 65°C. RNA was purified with the RNeasy^® Mini Kit (Qiagen, Hilden, Germany) before circularization, which was performed using T4 RNA ligase 1 (New England Biolabs, Ipswich, USA), 10% PEG 8000, 50 μM ATP and RNAseOut (Thermo Fisher Scientific, Waltham, USA). cDNA was synthesized using the RevertAid first strand cDNA kit (Thermo Fisher Scientific, Madison, USA) with a specific primer located near the 5′end of the GFP. The region containing the poly(A) was PCR amplified using nested primers (30 cycles reaction) and cloned into pGem^®-T Easy (Promega, Madison, USA). Individual clones were sequenced by Sanger. Sequencing results are provided in Supplementary Material (Data sheet S3 and S4). Annotation of the poly(A) site considered the first nucleotide of the terminator as position “1.” Sequence of all primers used in this experiment can be found in Supplementary Table 1.

de Felippes F.F., Shand K, & Waterhouse P.M. (2022). Identification of a Transferrable Terminator Element That Inhibits Small RNA Production and Improves Transgene Expression Levels. Frontiers in Plant Science, 13, 877793.

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In Vitro Circular RNA Synthesis

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RNAs were transcribed from annealed DNA-oligonucleotide templates (see Supplementary Table S1), using the HighScribe™ T7 high-yield RNA synthesis kit (NEB) in the presence of ATP, CTP, UTP, and GTP (each at 7.5 mM), GMP (30 mM GMP; Merck), and RNaseOut (Thermo Fisher Scientific) for 2 h at 37°C. The DNA template was digested by addition of RQ1 DNase (2 U per reaction, Promega), and incubation for 30 min at 37°C. Transcripts were purified using the Monarch RNA purification kit (NEB) and quantified by the Qubit™ RNA broad-range assay kit (Thermo Fisher Scientific).
For circularization, 60 μg transcribed RNA was incubated with 100 U of T4 RNA ligase (Thermo Fisher Scientific) in 1× T4 RNA ligase buffer, supplemented with 0.1 mg/ml BSA and RNaseOut (Thermo Fisher Scientific), overnight at 16°C in a final volume of 200 μl. RNA was recovered by phenol/chloroform extraction (Roth) and ethanol precipitation.
Gel purification was performed as described (33 ). To validate the circular conformation, 250 ng of gel-purified circular or linear RNA was incubated with or without 2 U of RNase R (Biozym; 30 min at 37°C). After digestion, 200 ng of RNAs were separated in a 10% denaturing polyacrylamide gel and visualized by ethidium bromide staining.

Pfafenrot C., Schneider T., Müller C., Hung L.H., Schreiner S., Ziebuhr J, & Bindereif A. (2021). Inhibition of SARS-CoV-2 coronavirus proliferation by designer antisense-circRNAs. Nucleic Acids Research, 49(21), 12502-12516.

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In Vitro RNA Deamination Assay

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An assay for RNA deamination was adapted for use with recombinant proteins from the poisoned primer extension design previously proposed (60 (link)). Purified recombinant MBP–APO1 and A1CF (residues 1–391) were combined at a 1:1 ratio and allowed to react on the in vitro transcribed APOB RNA described earlier. The reaction buffer consisted of 20 mM HEPES, pH 7.5, 50 mM NaCl, 5 mM DTT, 1 U/μl of RNAseOUT (Thermo Fisher) and 1 μM RNA. Ten-microliter reactions were allowed to incubate at 37°C for 1 h followed by the addition of 1.2 μM final of a 5′-FAM-labeled primer that binds downstream of the editing site with the sequence 5′-AAT CAT GTA AAT CAT AAC TAT CTT TAA TAT ACT GA-3′ and a denaturation step of 95°C for 10 min and subsequent step down to room temperature that stops the reaction, denatures the RNA strand and allows the primer to anneal. A reverse transcription buffer was then added that results in a final concentration of 2.5 U/μl Protoscript II (NEB), 1× manufacturer-recommended reverse transcription buffer, 5 mM DTT, 250 μM dTTP, dCTP and dATP, and 250 mM ddGTP (Tri-link Bio), and 0.1 U/μl of RNAseOUT (Thermo Fisher). After reverse transcription, the resulting products were run on a 20% acrylamide urea denaturing gel similar to DNA deamination.

Wolfe A.D., Li S., Goedderz C, & Chen X.S. (2020). The structure of APOBEC1 and insights into its RNA and DNA substrate selectivity. NAR Cancer, 2(4), zcaa027.

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EMSA Assay for m6A-RNA Binding

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Electrophoretic mobility shift assay was performed as previously described (Alarcón et al., 2015). The m⁶A-modified or -unmodified RNA probe was synthesized by Thermo Scientific with the sequence of 5'-CGAUCCUCGGCCAGGXCCAGCCUUCCCCA-3' (X=A or m⁶A). The capped or non-capped mRNA (Hsp70-5'UTR) was synthesized using the mMessage mMachine T7 Ultra kit (Ambion). The probe was labeled in a 50ul reaction mixture containing 2 µl RNA probe (1 µM), 5 µl 10×T4 PNK buffer (NEB), 1 µl T4 PNK (NEB), 40 U ml⁻¹ RNaseOUT (Thermo Scientific), 1 µl [³²P]ATP and 40 µl RNase-free water at 37°C for 1 h. The labeled probe was then purified by RNase-free micro bio-spin columns with bio-gel P30 (Bio-Rad 732-6250) according to manufacturer's protocols. After adding 2.5 µl 20×SSC (Promega) buffer, the RNA was denatured at 65 °C for 10 min and slowly cooled down. The purified probe (20 fmol) was incubated with increasing amount of GST-METTL3 at 4 °C for 1 h in binding buffer containing 10 mM HEPES, pH 8.0, 50 mM KCl, 1 mM EDTA, 0.05% Triton-X-100, 5% glycerol, 10 µg ml⁻¹ salmon DNA, 1 mM DTT and 40 U ml-1 RNaseOUT (Thermo Scientific). The RNA-protein mixture was loaded on Novex 8% TBE gel and run at 100 V at 4 °C. The signal was recoded via autoradiography.

Coots R.A., Liu X.M., Mao Y., Dong L., Zhou J., Wan J., Zhang X, & Qian S.B. (2017). m6A Facilitates eIF4F-Independent mRNA Translation. Molecular cell, 68(3), 504-514.e7.

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Antisense LNA Oligo Pull-Down Assay

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An antisense LNA oligo pull-down assay was adapted from (55 ,56 (link)) with modifications. Two antisense LNA oligos were designed to target the accessible regions within AtTR and labeled with Biotin at the 3′ end. About 2 g of formaldehyde-crosslinked four-day-old WT (WS) or attr seedlings were homogenized in lysis buffer (50 mM Tris-OAC pH 7.5, 10 mM EDTA, 1% SDS, 20 μl/ml Plant protease inhibitor mixture [Sigma-Aldrich], 1 μl/ml RNaseOUT [Thermo Fisher Scientific] and 6 mM DTT). The pre-warmed 2× hybridization buffer (50 mM Tris-OAC pH 7.5, 750 mM NaCl, 15% formamide, 1 mM EDTA, 20 μl/ml Plant protease inhibitor mixture [Sigma-Aldrich], 1 μl/ml RNaseOUT [Thermo Fisher Scientific] and 6 mM DTT) and 100 pmol antisense LNA oligos were incubated at 42°C for 2 h to achieve oligo annealing. About 100 μl Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher Scientific) was added and incubated for additional 45 min at 42°C. Beads were washed five times with wash buffer (2× SSC, 0.5% SDS, 1 mM PMSF, 5 mM DTT) before resuspending 90% of beads in SDS-loading buffer for protein analysis by western blot. The remaining 10% of beads was subjected to protease K digestion (10 mM Tris-Cl pH 7, 100 mM NaCl, 1 mM EDTA, 0.5% SDS and 5% 20 mg/ml protease K) at 50°C for 45 min prior to the RNA extraction by TRIzol reagent. The extracted RNA was analyzed by RT-qPCR for RNA abundance.

Song J., Castillo-González C., Ma Z, & Shippen D.E. (2021). Arabidopsis retains vertebrate-type telomerase accessory proteins via a plant-specific assembly. Nucleic Acids Research, 49(16), 9496-9507.

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Detecting Hepatitis E Virus in Wildlife

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The blood and stool samples were analyzed for HEV RNA in a one-step RT-qPCR assay targeting the ORF2/3 region of HEV1–HEV4 [17 (link),34 (link)]. Each 50-µL reaction mix contained 20 µL of extracted RNA, 25 µL of 2× universal master mix and 1 µL Superscript^® III Platinum One-Step Quantitative RT-PCR System with ROX (Invitrogen, Carlsbad, CA, USA), 40 U of RNaseOUT^™ (Invitrogen), and 0.2 µM of each primer and probe (Table 1).
Extracted nucleic acids from blood and fecal materials from moose, roe deer, red deer, and fallow deer were also analyzed for HEV RNA with a RT-qPCR assay, targeting the ORF2/3 region of moose HEV [34 (link)]. Each 50-µL reaction mix contained 10 µL of the extracted nucleic acids, 6 µL of RNase-free H₂O (Sigma, Saint Louis, MO, USA), 25 µL of 2× universal master mix and 1 µL of Superscript^® III Platinum One-Step Quantitative RT-PCR System with ROX (Invitrogen), 40 U of RNaseOUT^TM (Invitrogen), 0.6 µM of each primer, and 0.2 µM of probe (Table 1).
The cycling conditions for both RT-qPCR assays were as follows: 50 °C for 30 min, 95 °C for 10 min, followed by two-step cycling 45 times at 95 °C for 15 s and 60 °C for 60 s. All RT-qPCR reactions were carried out on a 96-well plate on an ABI 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA).

Roth A., Lin J., Magnius L., Karlsson M., Belák S., Widén F, & Norder H. (2016). Markers for Ongoing or Previous Hepatitis E Virus Infection Are as Common in Wild Ungulates as in Humans in Sweden. Viruses, 8(9), 259.

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PolyA+ RNA Extraction from Worms

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PolyA⁺ RNA was extracted from eggs, from 200 adult male worms and from 200 adult female worms using two rounds of the FastTrack MAG Maxi mRNA Isolation Kit (Invitrogen, Thermo Scientific, USA), as described [6 (link)], with the following modifications: 1,400 units of RNase Out (Invitrogen) and 20 mM of Vanadyl Ribonucleoside Complexes (VRC) were added to Lysis Buffer L4 at the first step of the sample preparation; at the final binding step, an additional 1,400 units of RNase Out (Invitrogen) was added to the sample while the tube remained in the rotator; treatment with 30 units of DNase I Amplification Grade (Invitrogen) was performed for 45 min at room temperature; and six washings were performed at the final washing step before elution. These modifications resulted in PolyA+ RNA samples with small percentages of rRNA contamination (3 to 7%), as estimated with a Bioanalyzer using the RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA).

Anderson L., Amaral M.S., Beckedorff F., Silva L.F., Dazzani B., Oliveira K.C., Almeida G.T., Gomes M.R., Pires D.S., Setubal J.C., DeMarco R, & Verjovski-Almeida S. (2015). Schistosoma mansoni Egg, Adult Male and Female Comparative Gene Expression Analysis and Identification of Novel Genes by RNA-Seq. PLoS Neglected Tropical Diseases, 9(12), e0004334.

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