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.
Superase rnase inhibitor
Manufactured by Thermo Fisher Scientific
Sourced in United States
SUPERase RNase inhibitor is a laboratory reagent designed to protect RNA samples from degradation by RNase enzymes. It functions by inhibiting the activity of RNase enzymes, thereby preserving the integrity of RNA molecules during various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Turbo dnase, by Thermo Fisher Scientific (6 mentions)
Cleancap egfp mrna, by TriLink (3 mentions)
Solution 13, by ChemoMetec (3 mentions)
A8 slide, by ChemoMetec (3 mentions)
Nucleocounter nc 3000, by ChemoMetec (3 mentions)
Cycloheximide (chx), by Merck Group (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Rnase 1, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Sw41ti rotor, by Beckman Coulter (2 mentions)
Purexpress, by New England Biolabs (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Trizol ls, by Thermo Fisher Scientific (2 mentions)
Superscript 4 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Sybr green 1 master mix, by Thermo Fisher Scientific (2 mentions)
Affinityscript rt buffer, by Agilent Technologies (1 mentions)
Dntp mix, by Thermo Fisher Scientific (1 mentions)
31 protocols using superase rnase inhibitor
1
Ribosome Profiling of Caenorhabditis elegans
2
HIV-1 pol Gene cDNA Synthesis
3
Cell-free Expression of Csx30-3 Protein
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3xHA tagged forms of Csx30–3 were cloned into pCDNA3.1 vectors and amplified by PCR using oligos containing the T7 promoter and terminator. Cell-free transcription-translation was performed using PURExpress (New England Biolabs) in 5 μL reactions containing 2 μL buffer A, 1.5 μL buffer B, 0.25 μL of Superase RNAse Inhibitor (Invitrogen), and 50–100 ng of PCR template. Reactions were incubated for 2 h at 37°C and directly transferred to in vitro reactions.
4
Efficient mRNA Delivery into NK Cells
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Before transfection, devices were passivated with NK MACS medium. NK cells were washed with DPBS, and prepared in NK MACS medium with 1:1000 Superase RNase inhibitor (Invitrogen), at a concentration of 2 million cells/ml. CleanCap eGFP mRNA (TriLink) was added to the cells with a concentration ranging from 20 to 80 µg/ml. Unless stated otherwise, the mix of NK cells and payload was flowed through a microfluidic device of 3.5 µm gap size device at a flow rate of 800 µl/min, and an mRNA concentration of 60 µg/ml. 250 µl of sample were processed per channel, and plated in a 24-well plate already containing 250 µl of 2× complete NK medium. Cell counts were performed after VECT transfection with NucleoCounter NC-3000 (ChemoMetec), using A8 slides (ChemoMetec) and solution 13 (ChemoMetec) as per manufacturer instructions. Cells were placed in the incubator for cell culture.
5
Efficient HSPC Transfection via VECT
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VECT medium (X-VIVO 10 Media (Lonza), and 1:1000 Superase RNase inhibitor (Invitrogen)) and complete HSPC medium (see Thawing of CD34+ HSPCs) were prepared. VECT microfluidic devices were passivated with 0.5 ml VECT medium per channel. HSPCs were spun at 300g for 5 min, washed with nuclease-free DPBS, and placed in HSPC VECT medium at 2 million cells/ml. Before VECT, CleanCap eGFP mRNA (TriLink) was added to the cells to achieve a final concentration of 160 µg/ml. For each sample, 200 µl HSPCs were flown through a 4 µm gap size device at a rate of 800 µl/min, into another 200 µl of pre-incubated 2× complete HSPC medium. Cell counts were performed after VECT transfection with NucleoCounter NC-3000 (ChemoMetec), using A8 slides (ChemoMetec) and solution 13 (ChemoMetec) as per manufacturer instructions. Lastly, samples were incubated at 37 °C, 5% CO2for 24 h before analysis.
6
Microfluidic Delivery of mRNA to T Cells
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Activated T cells were flown through a microfluidic device at a flow rate of 400 µl/min. Microfluidic devices were first passivated with TexMACS (Miltenyi Biotec). T cells were spun down at 300G for 5 min and washed with PBS. Cells were resuspended between 2 and 3 million cells/ml in T cell VECT Buffer: TexMACS (Miltenyi Biotec), and 1:1,000 Superase RNase inhibitor (Invitrogen). Before VECT transfection, CleanCap eGFP mRNA (TriLink) payload was added to the cells. 250 µl of reaction were processed per channel and plated in a 24-well plate already containing 250 µl complete T cell medium. Unless stated otherwise, the mix of T cells and payload was flowed through a microfluidic device of 4 µm gap size device at a flow rate of 800 µl/min, and an mRNA concentration of 70 µg/ml. Cell counts were performed after VECT transfection with NucleoCounter NC-3000 (ChemoMetec), using A8 slides (ChemoMetec) and solution 13 (ChemoMetec) as per manufacturer instructions.
7
Enteroid RNA Isolation and Gene Expression
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RNA was isolated from two pooled enteroid monolayers using TRIzol (Invitrogen) and the Direct-zol miniprep (Zymo Research) according to manufacturer’s protocol. Genomic DNA was removed with the TURBO DNA free kit (Invitrogen) in presence of SUPERase RNAse inhibitor (Invitrogen). cDNA was made with the Superscript IV first strand synthesis kit (Invitrogen) using oligo(dT)20 and random hexamers. Gene expression was assessed by PCR using the cDNA as template, the primers listed in the supplemental material (Table S5 ) and the GO Taq PCR kit (Promega). Amplicons were visualized on a 1% agarose gel.
8
RNA-Protein Binding Assay Protocol
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RNA-protein binding assays were carried out by following the method described by Satoh et al. (8 (link)) with slight modifications. Exponentially growing cells (4 × 108 total cells) expressing N-terminally glutathione S‐transferase (GST)-tagged wild-type or mutated Rnc1 proteins were disrupted in 500 μl extraction buffer (30 mM Tris HCl, pH 8, 1% Triton X-100, 2 mM EDTA, 1 mM dithiothreitol [DTT], plus protease and phosphatase inhibitor cocktails [obtained from Sigma-Aldrich and Roche Molecular Biochemicals, respectively]). Glutathione-Sepharose 4B (GE Healthcare, USA) was added to the cleared extracts, which were incubated for 2 h at 4°C. Sepharose was washed seven times in wash buffer (30 mM Tris HCl, pH 8, 1% Triton X-100, 2 mM EDTA, 1 mM DTT, 3 M NaCl plus phosphatase inhibitor cocktail) and two times in binding buffer (30 mM Tris HCl, pH 8, 1% Triton X-100, and 1 mM DTT). Fission yeast total RNA (100 μg) and 100 U ml−1 SUPERase RNase inhibitor (Invitrogen) were added to the washed Sepharose containing equivalent amounts of the purified GST-Rnc1 fusion and incubated for 2 h at 4°C. After two washes in binding buffer, the RNA bound to Sepharose was extracted with the RNeasy minikit (Qiagen, Germany). cDNA synthesis (10 μl RNA) and qPCR were performed as described above.
9
Total RNA Extraction and Purification
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Total RNA was extracted from 50 mg of bulk tissue using Trizol reagent according to manufacturer’s instructions (Ambion). To remove any residual DNA, RNA samples were treated with DNAse I (Ambion) using 2 U every 10 μg of RNA added with SUPERaseRNAse inhibitor (Invitrogen) in a reaction volume of 50 μL and incubated at 37°C for 30 min. After treatment, enzyme was inactivated and RNA was purified using Cleanup RNeasy® Mini kit according to manufacturer’s instructions (QIAGEN). The RNA concentration and purity were determined by spectrophotometric measurement using NanoDrop 2000 (Thermo Fisher Scientific) and by running denaturing formaldehyde agarose gel.
10
RNA Isolation and qPCR Analysis of Neurotransmitter Receptors
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