Cells were washed with cold HBSS, scrape-harvested directly into lysis buffer (10 mM HEPES pH 7.5, 125 mM KCl, 5 mM MgCl2, 1 mM DTT, 100 μg/mL cycloheximide, 100 μg/mL heparin, 1% NP40 made in DEPC-treated water), supplemented with RNasin Plus inhibitor (Promega) and HALT phosphatase and protease inhibitors (Thermo Scientific). Lysates were rotated at 4°C for 15 min, cleared by centrifugation for 10 min at 12,000 g, and supernatants loaded on pre-formed 17.5-50% sucrose gradients made in gradient buffer (10 mM HEPES pH 7.5, 125 mM KCl, 5 mM MgCl2, 1 mM DTT). Samples were centrifuged in a Beckman SW140 Ti rotor for 2.5h at 35,000 rpm, then eluted using a Brandel bottom-piercing apparatus connected to an ISCO UV monitor, which measured the eluate at OD 254.
Rnasin plus inhibitor
Manufactured by Promega
Sourced in United States
RNasin Plus is an RNase inhibitor that protects RNA from degradation by RNase enzymes. It is a recombinant protein that inhibits a broad range of RNase activities. RNasin Plus can be used in various RNA-related applications to maintain RNA integrity.
Automatically generated - may contain errors
Lab products found in correlation
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (7 mentions)
Sw140 ti rotor, by Brandel Inc (6 mentions)
Bottom piercing apparatus, by Teledyne (6 mentions)
Uv monitor, by Teledyne (6 mentions)
Ethylene glycol tetraacetic acid (egta), by Thermo Fisher Scientific (2 mentions)
Hoechst, by BD (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Mgcl2, by Merck Group (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Anti flag clone m2, by Merck Group (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Anti m6a, by Synaptic Systems (1 mentions)
Sw55ti rotor, by Beckman Coulter (1 mentions)
Facsaria fusion instrument, by BD (1 mentions)
Facsaria fusion, by BD (1 mentions)
11 protocols using rnasin plus inhibitor
1
Ribosome Profiling from Cell Lysates
2
FLAG-RIP and m6A-IP Protocols in Drosophila
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The FLAG-RIP and MeRIP protocols were performed using previously described protocols (Bienkowski et al., 2017 (link)) and (Lence et al., 2016 (link)) with some modification. Briefly, three replicates of 30 newly eclosed female flies were collected in 1.5 mL Eppendorf tubes and frozen in dry ice. Heads were removed with a 5.5 Dumont tweezer and homogenized with a mortar/pestle in Isolation buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA, 150 mM NaCl, and 1% SDS, 50 mM NaCl). This preparation was diluted into IP buffer (50 mM HEPES, 150 mM NaCl, 5 mM EDTA, 0.5 mM DTT, 0.1% NP-40) supplemented with protease inhibitors (Roche) and RNasin Plus Inhibitor (Promega). Lysates were incubated with anti-Flag (M2 clone; Sigma) or anti-m6A (Synaptic Systems) antibody and recovered on magnetic Protein G Dynabeads (Invitrogen). After overnight incubation at 4°C with rocking, beads were washed 5× in IP buffer and RNA was isolated from antibody-bead precipitates, or controls (input samples) using TRIzol (Thermo Fisher). Samples were treated with DNase I and RNA was purified using RNeasy Kit (QIAGEN).
3
Sucrose Gradient Fractionation of Cellular Lysates
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Cells were washed with cold HBSS, scrape-harvested directly into lysis buffer (10 mM Hepes, pH 7.5, 125 mM KCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml cycloheximide, 100 μg/ml heparin, and 1% NP-40), and supplemented with RNAsin Plus inhibitor (Promega) and HALT phosphatase and protease inhibitors (Thermo). Lysates were rotated at 4 °C for 15 min, cleared by centrifugation for 10 min at 12,000 g, and supernatants were loaded on preformed 17.5–50% sucrose gradients made in gradient buffer (10 mM Hepes, pH 7.5, 125 mM KCl, 5 mM MgCl2, and 1 mM DTT). Samples were centrifuged in a Beckman SW140 Ti rotor for 2.5 h at 35,000 rpm and then eluted using a Brandel bottom-piercing apparatus connected to an ISCO UV monitor, which measured the eluate at OD 254 nm.
4
Sucrose Gradient Ribosome Purification
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Cells were washed with cold HBSS, scrape-harvested directly into lysis buffer (10 mM Hepes, pH 7.5, 125 mM KCl, 5 mM MgCl2, 1 mM DTT, 100 µg/ml cycloheximide, 100 µg/ml heparin, and 1% NP-40 made in DEPC-treated water), and supplemented with RNAsin Plus inhibitor (Promega) and HALT phosphatase and protease inhibitors (Thermo Fisher Scientific). Lysates were rotated at 4°C for 15 min, cleared by centrifugation for 10 min at 12,000 g, and supernatants were loaded on preformed 17.5–50% sucrose gradients made in gradient buffer (10 mM Hepes, pH 7.5, 125 mM KCl, 5 mM MgCl2, and 1 mM DTT). Samples were centrifuged in a Beckman SW140 Ti rotor for 2.5 h at 35,000 rpm and then eluted using a Brandel bottom-piercing apparatus connected to an ISCO UV monitor, which measured the eluate at OD 254.
5
Sucrose Density Gradient Fractionation
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Cells were washed with cold HBSS, scrape-harvested directly into lysis buffer (10 mM HEPES pH 7.5, 125 mM KCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml cycloheximide, 100 μg/ml heparin, 1% NP40 made in DEPC-treated water), supplemented with RNasin Plus inhibitor (Promega) and HALT phosphatase and protease inhibitors (Thermo Scientific). Lysates were rotated at 4 °C for 15 min, cleared by centrifugation for 10 min at 12,000 g, and supernatants loaded on pre-formed 17.5–50% sucrose gradients made in gradient buffer (10 mM HEPES pH 7.5, 125 mM KCl, 5 mM MgCl2, 1 mM DTT). Samples were centrifuged in a Beckman SW140 Ti rotor for 2.5 h at 35,000 rpm, then eluted using a Brandel bottom-piercing apparatus connected to an ISCO UV monitor, which measured the eluate at OD 254.
6
Polysome Fractionation for RNA Analysis
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Cells were treated with 100 μg/ml cycloheximide for 10 min, washed with HBSS, and harvested into lysis buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml cycloheximide, 1% Triton-X100 in DEPC-treated water) supplemented with RNasin Plus inhibitor (Promega, Madison, WI USA) and HALT phosphatase and protease inhibitors (Thermo Scientific, Waltham, MA, USA). Lysates were rotated at 4 °C for 10 min, and centrifuged for 10 min at 10,000×g. Supernatants were loaded onto 10–50 % sucrose gradients made in gradient buffer (150 mM NaCl, 20 mM Tris [pH 7.4], 5 mM MgCl2, 1 mM DTT, 100 µM cycloheximide, 0.25% NP40, RNasin) and centrifuged in a Beckman SW55Tirotor for 1.5 h, 45,000xg at 4 °C. Samples were eluted using a Brandel bottom-piercing apparatus attached to a syringe pump. An ISCO UV monitor was used to measure the eluate at OD 254.
7
In Vitro DICER Cleavage Assay
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In vitro DICER cleavage assays were done in a 45 µL reaction mixture containing 20 mM PIPES (pH 6.2), 1.5 mM MgCl2, 80 mM NaCl, 1 mM dithiothreitol (DTT), 0.05% NP40, 10% glycerol, 3U/µL RNasin plus inhibitor (Promega), 10 mM EGTA, 1.5 nM DICER, and 0.15 nM substrate RNA. Reaction mixtures were incubated at 37°C, and 7.5 µL aliquots were taken after 0, 5, 10, 30, and 60 min. The reactions were stopped by adding an equal volume of gel loading buffer (80% formamide, 20% glycerol, 0.025% BPB). After heating at 80°C for 10 min, samples were analyzed by 10% Urea-PAGE and quantified using Molecular Dynamics and ImageJ (Ota et al. 2013 (link)).
8
Sucrose Density Gradient Fractionation
9
Isolation and Purification of Cardiac Nuclei
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Cardiac tissues were thawed on ice and cut into small pieces. Minced tissue was pre-digested with a 5-ml enzyme solution of collagenase (2500 U, Thermo Fisher Scientific) in HBSS + /+ (Gibco) for 10 min at 37 °C in a water bath. After centrifugation at 500 g and 4 °C for 5 min, the supernatant was discarded, and nuclei were isolated after cell disruption with a glass dounce homogenizer (five strokes with a loose pestle and ten strokes with a tight pestle). After filtering (20-µm strainer, pluriSelect), the suspension was centrifuged at 1000 g and 4 °C for 6 min and resuspended in 500 µl staining buffer containing 1% BSA (Sigma-Aldrich), 5 nM MgCl2 (Sigma-Aldrich), 1 mM EDTA (Gibco), 1 mM EGTA (Gibco), 0.2 U µl−1 RNasin Plus Inhibitor (Promega) and 0.1 µg ml−1 Hoechst (Life Technologies) in Dulbecco´s phosphate buffered saline (DPBS). Hoechst-positive nuclei were separated from cell debris by using the FACSAria Fusion instrument (BD Biosciences) and sorted into a staining buffer without Hoechst at 4 °C.
10
Cardiac Nuclei Isolation for snRNA-seq
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Prior to snRNA-seq, single nuclei were isolated from left ventricular tissue samples. Cardiac tissues were thawed on ice and cut into small pieces. Minced tissue was then pre-digested in 5 ml enzyme solution of collagenase (2500 U,Thermo Fisher Scientific) in HBSS+/+ (Gibco) for 10 min at 37 • C in a water bath. After centrifugation at 500 ×g and 4 • C for 5 min, the supernatant was discarded and nuclei were isolated after cell disruption with a glass Dounce homogenizer according to the previously published protocol [12] . After homogenization of the tissue and filtering through a 20 μm strainer (pluriSelect), the nuclei suspension was centrifuged at 1000 ×g at 4 • C for 6 min and resuspended in 500 μl staining buffer containing 1% BSA (Sigma Aldrich), 5 nM MgCl2 (Sigma Aldrich), 1 mM EDTA (Gibco), 1 mM EGTA (Gibco), 0.2 U/μl RNasin Plus Inhibitor (Promega) and 1:100,000 Hoechst (Life Technologies) in PBS. Then, Hoechst-positive single nuclei were separated from cell debris by FACS using the FACS ARIA Fusion instrument (BD Biosciences) and sorted into staining buffer at 4 • C.
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