Superase in

Manufactured by Thermo Fisher Scientific

Sourced in United States

Superase-In is a ribonuclease inhibitor that protects RNA from degradation during sample preparation and analysis. It is a concentrated, recombinant, and animal-free product designed for use in sensitive RNA applications.

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Close spelling variants of this product

SUPERase•InTM RNase Inhibitor RNase SUPERase-In SUPERase•In Inhibitor SUPERase-In ribonuclease inhibitor SUPERase•In™ RNAase inhibitor SUPERase-In RNase Inhibitor SUPERase

291 protocols using superase in

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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Saliva RNA Extraction and Preservation

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Saliva samples were processed immediately after collection according to a previously published method (35 (link)). Briefly, the saliva samples were centrifuged at 2,600g for 15 minutes, at 4°C. The supernatant was separated from the pellet, and the RNase inhibitor (Superase-In, Ambion Inc., Austin, TX) was added to the supernatant-- 5ul Superase-In/ml of supernatant. All samples were stored at −80 °C in aliquots until further use.

Cheng Y.S., Jordan L., Chen H.S., Kang D., Oxford L., Plemons J., Parks H, & Rees T. (2016). Chronic Periodontitis Can Affect the Levels of Potential Oral Cancer Salivary mRNA Biomarkers. Journal of periodontal research, 52(3), 428-437.

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Mitochondrial Purification and Decontamination

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Briefly, mitochondria were purified from 4–6 × 10⁷ cells using a mitochondria isolation kit for cultured cells (Thermo Fisher Scientific) according to manufacturer instructions; all buffers were supplemented with 60 U ml⁻¹ SUPERase-In (Ambion) and 1× Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (Life Technologies). Mitoplasts were obtained by incubating purified mitochondria in RNase A–containing hypotonic buffer (Hepes, pH 7.2, supplemented with 1× Halt Protease and Phosphatase Inhibitor Single-Use Cocktail and 10 µg/ml RNase A [Roche]) for 20 min on ice and subsequently incubated for 10 additional min at room temperature in order to remove all possible cytosolic RNA contaminants. The purified mitoplasts were then washed three times with mitoplast isolation buffer (250 nM mannitol, 5 mM Hepes, pH 7.2, 0.5 mM EGTA, 1 mg/ml BSA supplemented with 60 U ml⁻¹ SUPERase-In [Ambion] and 1× Halt Protease and Phosphatase Inhibitor Single-Use Cocktail).

Vendramin R., Katopodi V., Cinque S., Konnova A., Knezevic Z., Adnane S., Verheyden Y., Karras P., Demesmaeker E., Bosisio F.M., Kucera L., Rozman J., Gladwyn-Ng I., Rizzotto L., Dassi E., Millevoi S., Bechter O., Marine J.C, & Leucci E. (2021). Activation of the integrated stress response confers vulnerability to mitoribosome-targeting antibiotics in melanoma. The Journal of Experimental Medicine, 218(9), e20210571.

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Mitoplast Isolation from Cultured Cells

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Briefly, mitochondria were purified from 4-6 ´ 10 7 cells using mitochondria isolation kit for cultured cells (Thermo Fisher Scientific) according to manufacturer instructions, all buffers were supplemented with 60 U mL -1 Superase-In (Ambion) and 1´ Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (Life Technologies).
Mitoplasts were obtained by incubating purified mitochondria in RNase A-containing hypotonic buffer (HEPES pH 7.2 supplemented with 1´ Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (Life Technologies) and 10 μg mL -1 RNase A (Roche)) for 20 min on ice and subsequently incubated for 10 additional min at room temperature in order to remove all possible cytosolic RNA contaminants. The purified mitoplasts were then washed thrice with Mitoplast Isolation Buffer (MIB: 250 nM Mannitol, 5 mM HEPES pH 7.2, 0.5 mM EGTA, 1 mg mL -1 BSA supplemented with 60 U mL -1 Superase-In (Ambion) and 1´ Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (Life Technologies)).

Vendramin R., Konnova A., Adnane S., Cinque S., Katopodi V., Knezevic Z., Karras P., Demesmaeker E., Bosisio F.M., Rizzotto L., Bechter O., Marine J., & Leucci E. (2020). Activation of the Integrated Stress Response in drug-tolerant melanoma cells confers vulnerability to mitoribosome-targeting antibiotics.

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Affinity Purification of miRNA-Target Complexes

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3×10⁶ cells were seeded into each 10 cm dish one day before transfection. 3μg of miRNA expression vector and 2 μg of target expression vector were transfected into the cells by PolyJet. Cells were washed with PBS once and then collected by using cell scraper in 600 μL modRIPA buffer supplemented with proteinase inhibitors cocktail (Roche) and Superase-in (ThermoFisher Scientific) at 1:200. 10 pmol of biotinylated probes (Supplementary Table S1) were incubated with cell lysates in the hybridization buffer (70mM Tris-Cl pH7.0, 675mM Nacl, 5.5 mM EDTA, 1.45% SDS, 15% formamide) with proteinase inhibitors cocktail (Roche) and Superase-in (ThermoFisher Scientific), for 4h with rotating. 30 μL C1 streptavidin Dynabeads (ThermoFisher Scientific) were incubated with each sample at room temperature for 1h. After washed for 3 times with 1ml wash buffer (2x SSC, 0.5% SDS), RNAs associated with Dynabeads were extracted by Trizol.

Yang A., Ros X.B., Shao T.J., Jiang M., Li K., Villanueva P., Dai L, & Gu S. (2019). 3’ Uridylation Confers miRNAs With Non-canonical Target Repertoires. Molecular cell, 75(3), 511-522.e4.

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Single-Cell RNA-Seq of Apoptotic and Blast Cells

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KG1a cells were stained using the PE Annexin V Apoptosis Detection Kit (BD Life Science, catalog # 559763). Live cells (Annexin V^-/7-AAD^-) were sorted into individual wells of a 96 well plate containing lysis buffer 2.5μL RLT Plus Lysis Buffer (QIAGEN, catalog # 1053393) with 1U/μL SUPERase-In (ThermoFisher, catalog # AM2696). Before sorting, bulk KG1a samples of 1,000,000 cells were collected from both the untreated and treated populations for comparison with single cells. HL60 cells were stained with Propinium Iodide (PI) (ThermoFisher, catalog # P1304MP) and live cells (PI^-) were sorted into 96 well plate containing lysis buffer 2.5μL RLT Plus Lysis Buffer with 1U/μL SUPERase-In.
Cryopreserved primary human cells were resuspended in thawing media (IMDM, 20% FBS), washed twice and resuspended. The cells were then rested for 1 h at 37 °C before preparation for flow cytometry. Cells (1 × 10⁶/100 μl) were stained with 1.5 μg/mL propidium iodide (PI, Sigma-Aldrich, P1304MP), 1:20 CD45-PECy7 (2D1, Life Technologies, catalog # 25-9459-42), 1:20 CD33-FITC (WM-53, Life Technologies, catalog # 11-0338-42) and 1:20 CD19-BV711 (SJ25C1, BD Biosciences, catalog # 563036). Single blasts (PI⁻/CD45^dim) were collected in 2.5μL RLT Plus Lysis Buffer containing 1U/μL SUPERase-In in 96 well plates.

Hunt K.V., Burnard S.M., Roper E.A., Bond D.R., Dun M.D., Verrills N.M., Enjeti A.K, & Lee H.J. (2022). scTEM-seq: Single-cell analysis of transposable element methylation to link global epigenetic heterogeneity with transcriptional programs. Scientific Reports, 12, 5776.

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Ribosome Display Protocol for Protein Selection

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Ribosome display was performed using a custom PURExpress kit from New England Biolabs (NEB) that lacks release factors 1, 2 and 3, and T7 RNA polymerase. Specifically, we prepared a 200 µl master mix containing 80 µl of solution A, 60 µl of solution B, 4 µl of disulfide enhancers 1 and 2 (E6820S; NEB) (if required) and 4 µl of Superase In (AM2696; Thermo Fisher). We then injected the master mix into each lane of the flow cell, being careful to avoid the introduction of bubbles, before incubating the flow cell at 37 °C for 60 minutes. Once the incubation period was complete, we cooled the flow cell down to 20 °C, before washing and stabilizing the ribosomes with ribosome display buffer (50 mM Tris(hydroxymethyl)aminomethane acetate, 150 mM NaCl, 50 mM magnesium acetate, 0.1% Tween 20 and 1 U ml⁻¹ of Superase In (AM2696; Thermo Fisher), pH 7.5).
With the ribosomes stabilized by the display buffer, we block the flow cell with binding buffer (ribosome display buffer with 0.1% BSA; A9647; Sigma-Aldrich). After flow-cell blocking, we image the surface to determine a baseline for background fluorescence.

Porebski B.T., Balmforth M., Browne G., Riley A., Jamali K., Fürst M.J., Velic M., Buchanan A., Minter R., Vaughan T, & Holliger P. (2023). Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening. Nature Biomedical Engineering, 8(3), 214-232.

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Cellular Fractionation and RNA Extraction

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Fractionation of cytosolic and nuclear extracts was performed using the NER-PER Nuclear and Cytoplasmic Extraction Reagents Kit (ThermoFisher) according to the manufacturer’s instructions, with the exception that 2 μl SUPERase In (Invitrogen) were added to CER I and 1 μl SUPERase In (Invitrogen) were added to CER II. RNA from these fractions was isolated using Trizol (Sigma-Aldrich) according to the manufacturer’s instructions, and was subsequently used for RT-qPCR.

Tai-Schmiedel J., Karniely S., Lau B., Ezra A., Eliyahu E., Nachshon A., Kerr K., Suárez N., Schwartz M., Davison A.J, & Stern-Ginossar N. (2020). Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication. PLoS Pathogens, 16(4), e1008390.

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Selective Enrichment of ZIKV-Derived RNA

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RNA was fragmented to an average size of 100 nucleotides using RNase III (Ambion) and was cleaned by RNA clean & concentrator (Zymo Research). Copper-free Click reaction was carried at 37 °C for 90 minutes in the presence of 150 μM Click-IT Biotin DIBO Alkyne (Life technologies) and 0.5 units/μl Superase-In (Invitrogen). Reaction was terminated by RNA clean & concentrator (Zymo Research). Biotinylated RNA was pulldown using Dynabeads MyOne Streptavidin C1(Invitrogen) at the following reaction conditions: 100 mM Tris-Cl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween-20, 0.5 unit/μl Superase-In. Beads were captured on a magnet and were washed 5 times with 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 3.5M NaCl, 0.1% Tween-20. RNA was eluted by adding 95% Formamide, 10 mM EDTA solution and incubating at 65oC for 5 minutes. To avoid enrichment of small RNA chimeric reads that cannot be double-aligned to the reference ZIKV genome / Human transcriptome, RNA was size fractionated on 10% TBE-Urea gel and fragments corresponding to a size of 100-200 nucleotides were eluted overnight at 4 °C in 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl, 0.1% SDS. RNA was concentrated using RNA clean & concentrator (Zymo Research).

Ziv O., Gabryelska M.M., Lun A.T., Gebert L.F., Sheu-Gruttadauria J., Meredith L.W., Liu Z.Y., Kwok C.K., Qin C.F., Macrae I.J., Goodfellow I., Marioni J.C., Kudla G, & Miska E.A. (2018). COMRADES determines in vivo RNA structures and interactions. Nature methods, 15(10), 785-788.

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Selective Enrichment of ZIKV-Derived RNA

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