For RNAse I degradation experiment, 40 μl aliquots of DNA‐free RNA was incubated at 37°C for 40 min in the presence of increasing concentrations of RNAse I (Ambion): 0 (buffer only), 2, 10, 20 and 40 Units RNAse I μg−1 RNA. The reaction was stopped by adding 10 μl β‐mercaptoethanol and RNA was recovered by ethanol precipitation: 5 μl of 7.5 M ammonium acetate and 137.5 μl 100% ethanol was added and the mixture was precipitated overnight at −20°C. RNA was pelleted by centrifugation 16 000g for 40 min at 4°C and the pellet was washed with 480 μl ice cold 70% ethanol and pelleted by centrifugation at 16 000g for 30 min at 4°C. The pellet was air dried and re‐suspended in 40 μl of DEPC‐treated water. An aliquot of RNA that did not undergo ethanol precipitation was also included for comparison (designated NT: ‘Not Treated’).
Rnase 1
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, China, Canada
RNase I is an enzyme that cleaves single-stranded RNA molecules. It is widely used in molecular biology laboratories for various applications, such as the removal of RNA from DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Thermo Fisher Scientific (32 mentions)
Turbo dnase, by Thermo Fisher Scientific (26 mentions)
Superase in, by Thermo Fisher Scientific (15 mentions)
Superase in rnase inhibitor, by Thermo Fisher Scientific (12 mentions)
Cycloheximide (chx), by Merck Group (9 mentions)
Rnase a, by Thermo Fisher Scientific (8 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (8 mentions)
Rnase t1, by Thermo Fisher Scientific (7 mentions)
Proteinase k, by Thermo Fisher Scientific (7 mentions)
Sw41ti rotor, by Beckman Coulter (7 mentions)
Superscript 3, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Takara Bio (4 mentions)
Benzonase, by Merck Group (4 mentions)
Mirneasy mini kit, by Qiagen (4 mentions)
Rnaseout, by Thermo Fisher Scientific (4 mentions)
T4 pnk, by New England Biolabs (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Accuprime supermix 1, by Thermo Fisher Scientific (4 mentions)
Hiseq 2000, by Illumina (4 mentions)
Circligase, by Illumina (4 mentions)
213 protocols using rnase 1
1
RNase I Degradation of RNA
2
Selective Ribosome Removal with RNase I
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For selective removal of eukaryotic specific expansion segments, RNCs or nonprogrammed ribosomes (80S) were incubated with 40 U RNase I (Thermo Fisher) per 1 A260 unit of ribosomes for 45 min at 25°C. The reaction was stopped with 0.5 U SUPERase-In (Thermo Fisher) RNase Inhibitor per 1 U of RNase I, and the sample was placed on ice. The RNase I-treated 80S and RNCs are referred to as rt80S and rtRNC, respectively.
3
Identifying Nucleic Acid Bound to Rep
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To determine the identity of nucleic acid bound to Rep, we independently treated the purified sample with DNase I or RNase I (Thermo Fisher) according to the manufacturer’s protocol. For each reaction, a total of 40 μM purified Rep (10 μl) was first denatured at 90°C for 15 min. The sample was cooled to room temperature and then digested with 10 U of RNase I in 20 mM Tris-acetate (pH 8.0), 100 mM NaCl, and 0.1 mM EDTA or with 1 U of DNase I in 100 mM Tris-HCl (pH 7.5), 25 mM MgCl2, 1 mM CaCl2, and 50 mM EDTA for 2 h. A 1% agarose gel (40 ml) was cast in the presence of 1 μl of SYBR-Gold nucleic acid stain (10,000× concentrate in dimethyl sulfoxide [DMSO]; Thermo Fisher). For controls, a 1 μM concentration of a 12- and a 44-nt oligonucleotide (10 μl each) was also processed with the agarose gel. Gels were visualized using a UV-transilluminator at 302 nm.
4
Ribosome Profiling in HEK293 Cells
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HEK293 (H. sapiens, RRID: CVCL_0045) cells were transfected with a plasmid encoding GFP (pMAX_GFP, Lonza). GFP expression was monitored by fluorescence microscopy (Olimpus IX-50). After 24 h after transfection, cells were treated with harringtonine (2 μg/ml) for 3 min, followed by cycloheximide addition (CHX, 10 μg/ml, SIGMA cat. no. 01810) and incubation for 5 min at 37°C. Controls were not treated with harringtonine. Cell lysates were obtained using a hypotonic lysis buffer (IMMAGINA Biotechnology, cat. no. RL001-1). Lysate absorbance at 260 nm was measured by Nanodrop ND-1000 UV-VIS Spectrophotometer and then diluted to a final value of 1.7 a.u. (absorbance measured at 260 nm) in 250 μl of W-buffer (IMMAGINA Biotechnology, cat. no. RL001-4). Ribosome protected Fragments (RPFs) were generated by treating the diluted lysate with 12.7 U of RNase I (Ambion, cat. no. AM2295) at room temperature for 45 min in an orbital mixer (31 (link)). RNase I digestion was stopped by adding 10U of SupeRNase Inhibitor (Thermo Scientific, cat. no. AM2696) for 10 min on ice.
5
Ribosome Profiling of Caenorhabditis elegans
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Concentrated lysis buffer was added to each frozen sample to a final concentration of 20 mM Tris-HCl (pH = 7.4), 150 mM NaCl, 5mM MgCl2, 0.5X Protease Inhibitor (Sigma, Cat:P2714), 1 mM DTT, 0.1 mg/mL cycloheximide (Millipore, Cat:C4859), 1% (v/v) Triton X-100 and 5 U/mL Turbo DNase (Invitrogen, Cat:AM2238) (Ingolia et al., 2012 (link)), and worm pellets were kept on ice until fully thawed. Suspended worms were transferred to 400 μm silica beads tube (OPS Diagnostic, Cat: PFAW-400-100-04) and lysed in bead beater homogenizer for 4 min at 4°C. Lysates were then centrifuged at 25,000 rcf for 10 min at 4°C, and supernatants were collected. To generate monosomes, RNase I (Invitrogen, Cat: AM2294) was added to a final concentration of 0.2 U per μl of harvested worm pellet. The digestion was incubated at room temperature for 40 min with gentle rotation and then quenched by adding SUPERase RNase Inhibitor (Invitrogen, Cat: AM2694) at 4 U per RNase I unit. The lysates were then loaded onto 5-40% (m/v) sucrose gradients prepared with lysis buffer without Triton X-100 and centrifuged at 32,000 rpm for 3 h at 4°C in an SW41Ti rotor (Beckman Coulter, Cat:331362). The sucrose gradients were fractionated using BR-188 Density Gradient Fractionation System with 60% (m/v) sucrose as chase solution, and monosome fractions were collected according to OD254 profiles.
6
RNA Digestion and Analysis
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RNA digestion of 20 OD260nm of cell extracts were performed by using 1 unit of Xrn1 (Biolabs) in NEB buffer 3 at 25 °C during 30 min unless otherwise indicated. NEB Buffer 3 was replaced by T4 PNK buffer (NEB) in kinase assays in the presence or absence of Xrn1 (Figs. 3 a and 5d ). For RNase I treatment of cell extracts, 20 OD260nm of extracts (prepared without heparin) were incubated with 0.5, 1, or 2 µl of RNase I (Invitrogen, 100 units/µl) 30 min at 25 °C. For total RNA treatment, 5 µg of RNA were digested 30 min at 25 °C. All RNase treatments were followed by RNA extraction and northern blot analysis as described above.
7
Ribosome Profiling of Caenorhabditis elegans
8
Cell Lysis and RNA Digestion Protocol
9
Cell Lysis and RNA Digestion Protocol
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Frozen cell pellet in a 50 ml tube was resuspended in 30 ml of PGB Cell Lysis Buffer Complete (PGB Cell Lysis Buffer supplemented with 1/1000 1M DTT, 1/1000 anti-RNase (Life Technologies, AM2690), 4/1000 Protease Inhibitor Cocktail Set III, EDTA-Free (MERCK, 539134), 0.1 μl / 1ml TURBO DNase (Life Technologies, AM2238)). The lysate was homogenized by passing twice through a syringe with a 21G needle and cleared by centrifugation at 14,000 ×g for 5 min at 4°C. The supernatant was collected and centrifuged again at 14,000 ×g for 15 min 4°C. The supernatant was collected and 20 ml of PGB Cell Lysis Buffer Complete was added and filtered through a 0.45 μm syringe filter. Unprotected RNA was digested by adding 0.4 U/ml (High RNase condition) or 0.2 U/ml (Low RNase, 2nd round ligation-minus control and STAU1 induction-minus condition) of RNase I (Life Technologies, AM2294) to the lysate, and the lysate was incubated for 5 min at 37°C. After incubation, the tube was transferred to ice for a minimum of 5 min. In order to stop RNase I activity, 20 μl of SUPERaseIn (Life Technologies, AM2694) was added.
10
Polysome Profiling of Cytosolic and Membrane Fractions
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Polysome profiling was performed from cytosolic and membrane fraction lysates. Lysates were thawed at 25 °C and cleared for 5 min at 12000 g. 4 A260 units of cytosolic fractions and 2-4 A260 units of membrane fraction were loaded onto linear 15 % −45 % sucrose gradients and centrifuged for 2 h at 273865 g at 4 °C in a Beckman SW41 rotor. Gradients were prepared by underlying 45% sucrose dissolved in polysome buffer 1.0 (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 100 mM KCl, 0.1 mg/ml CHX) a 15% sucrose solution and mixed using a Gradient Master. Lysates were treated with 20 U RNAse I (Ambion) per A260 unit of extract for 5 min at 25 °C. Digestion was stopped through the addition of 10 U RNAse Inhibitor SUPERase-In (Ambion) per 20 U of RNAse I. Treated extracts were loaded onto linear 10 % − 50 % sucrose gradients prepared in polysome buffer 2.0 (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 100 mM NH4Cl, 0.1 mg/ml CHX) and centrifuged for 3 h at 209,678 g at 4 °C in a Beckman SW41 rotor. Monosome peaks were collected and subjected to hot phenol RNA extraction in the presence of 1% SDS.
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