In order to recognize the skin layers of the biopsy, a fast hematoxylin and eosin staining was performed on the slices with slight modifications of the protocol published by Yee and colleagues [9 (link)]. Staining was performed after removing the glass slides from − 80 °C and leaving them at room temperature for few minutes to dry. Tissues were dehydrated in 70% ethanol (prepared in DEPC water) for 20 s. For the hematoxylin staining, 500 μl of diluted 1:2 hematoxylin solution in ethanol added with 1 U/μl RNasin® Plus RNase Inhibitor (Promega, Madison, WI, USA) shortly before the use [10 (link), 12 (link)], was used for 10 s. After a 10 s wash in DEPC water and an additional 10 s wash in 70% ethanol, the slices were stained for 2 s with 250 μl of diluted 1:2 eosin solution in ethanol containing 1 U/μl RNasin® Plus RNase Inhibitor (Promega, Madison, WI, USA) shortly before the use. Instead of staining the glass slides by immersion, they were horizontally positioned on the bench and drops of colored solutions were added over. Subsequently, after a 10 s wash in DEPC water and a 10 s wash in 70% ethanol, the tissues on glass slides were dried at room temperature for few minutes and then stored at − 80 °C before proceeding with the laser microdissection.
Rnasin plus rnase inhibitor
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RNasin Plus RNase Inhibitor is a recombinant ribonuclease (RNase) inhibitor protein that protects RNA from degradation by RNase enzymes. It is a potent and specific inhibitor of RNase A, RNase B, and RNase C.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (13 mentions)
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
M mlv reverse transcriptase, by Promega (7 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (7 mentions)
Turbo dnase, by Thermo Fisher Scientific (6 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Dntps, by Promega (5 mentions)
Fluorescence dissecting microscope, by Leica (4 mentions)
Dl dithiothreitol dtt, by Merck Group (4 mentions)
Neurotrace 500 525, by Thermo Fisher Scientific (4 mentions)
Needle blade micro knife, by Fine Science Tools (4 mentions)
Vibratome, by Leica (4 mentions)
Pierce phosphatase inhibitor, by Thermo Fisher Scientific (4 mentions)
Pierce protease inhibitor, by Thermo Fisher Scientific (4 mentions)
Igepal ca 630, by Merck Group (4 mentions)
Rq1 dnase, by Promega (4 mentions)
Superase in rnase inhibitor, by Thermo Fisher Scientific (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
187 protocols using rnasin plus rnase inhibitor
1
Rapid Hematoxylin and Eosin Staining for Skin Biopsy
2
Isolation of Nuclei for DroNc-seq Assay
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5-6 bilateral tissue dissections were placed in a dounce homogenizer with 1 mL cold (4 °C) Lysis Buffer containing 10 mM trisHCl pH 8 (Sigma-Aldrich), 250 mM Sucrose (Sigma-Aldrich), 25 mM KCl, 5 mM MgCl2 (Sigma-Aldrich), 0.1% Triton x100 (Sigma-Aldrich), 0.5% RNasin Plus RNase Inhibitor (Promega), 0.1 mM Dithiothreitol (DTT) (Sigma-Aldrich) in UltraPure™ DNase/RNase-Free Distilled Water (Thermo Fisher Scientific). After douncing for 20 times, the solution was filtered through a sterile 20 µm Cell Strainer (pluriSelect), collected in 1.5 ml DNA LoBind® Tubes (Eppendorf), and centrifuged for 10 min at 900 g (rcf) at 4 °C. The “slow sedimenting” component (debris and membranes) was aspirated and discarded while the “fast sedimenting” component (nuclear fraction) was gently resuspended in a 1 mL of Working Solution containing 1X pH 7.4 RNase free PBS (Thermo Fisher Scientific), 0.01% Albumin Bovine Serum (BSA) (Sigma-Aldrich), 0.5% RNasin Plus RNase Inhibitor (Promega) in UltraPure™ DNase/RNase-Free Distilled Water (Thermo Fisher Scientific). Nuclei were kept on ice while transferred to the BNORC Functional Genomics and Bioinformatics (FGB) Core for DroNc-seq assay.
3
RNA Fractionation by Soft Lysis
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For RNA fractionation was used soft lysis procedure [12 (link)]. The HEK293T cells were detached by treating with 1× Trypsin, transferred into 1.5 ml tube and centrifuged at RT 168 g for 5′. The pellet was lysed with 175 μl/106 cells of cold RLN1 solution (50 mM Tris HCl pH 8, 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40, RNasin Plus RNase Inhibitor, Promega) and incubated 5′ in ice. Next, the suspension was centrifuged at 4 °C 300 g for 2′ and the supernatant, corresponding to the cytoplasmic fraction, was transferred into a new tube and stored in ice. The pellet containing nuclei was extracted with 175 μl/106 cells of cold RLN2 solution (50 mM Tris HCl pH 8, 500 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40, RNasin Plus RNase Inhibitor, Promega) and 5′ incubated in ice. The suspension was centrifuged at 4 °C 16360 g for 2′ and the supernatant, corresponding to the nuclear-soluble fraction, was transferred into a new tube and stored in ice. The remaining pellet corresponds to the chromatin-associated fraction. The ratio of fractions to total RNA was estimated using RT-qPCR. All experiments were performed in triplicate.
4
Larval Brain Proteomic Analysis
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Third instar larval brains were dissected in Schneider’s insect medium and then flash frozen in liquid nitrogen. Brains were homogenised using a pestle in IP buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% NP-40, 10% glycerol, one mini tablet of Complete EDTA-free protease inhibitor and 2 μl RNAse inhibitor (RNAsin Plus RNase Inhibitor, Promega). RNA was extracted using the RNASpin Mini kit (GE Healthcare) according to manufacturer’s instructions.
Reverse transcription and quantitative PCR cDNA was produced from extracted RNA using RevertAid Premium Reverse Transcriptase (Thermo Fisher Scientific) according to manufacturer’s instructions with the addition of 1 μl RNAse inhibitor (RNAsin Plus RNase Inhibitor, Promega).
Real time quantitative PCR was performed using primers specific to a transcript of interest, and where possible spanning an exon junction. qPCR was performed using SYBR Green Master Mix with the CFX96 Touch Real-Time PCR Detection System (BioRad). Cycle threshold (C(T)) values were calculated from the BioRad CFX software using a second differential maximum method. Input samples were used for a dilution series and the percentage input of each gene was calculated in the IP samples as a measure of pulldown. For primer sequences seeTable 1 .
Reverse transcription and quantitative PCR cDNA was produced from extracted RNA using RevertAid Premium Reverse Transcriptase (Thermo Fisher Scientific) according to manufacturer’s instructions with the addition of 1 μl RNAse inhibitor (RNAsin Plus RNase Inhibitor, Promega).
Real time quantitative PCR was performed using primers specific to a transcript of interest, and where possible spanning an exon junction. qPCR was performed using SYBR Green Master Mix with the CFX96 Touch Real-Time PCR Detection System (BioRad). Cycle threshold (C(T)) values were calculated from the BioRad CFX software using a second differential maximum method. Input samples were used for a dilution series and the percentage input of each gene was calculated in the IP samples as a measure of pulldown. For primer sequences see
5
SARS-CoV-2 RNA Extraction from Cell Culture
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The MagneSil® Total RNA Mini Isolation System (Promega; Madison, WI; Cat. No. Z3351) was used for extraction of viral RNA from cell culture following the manufacturer’s protocol; however, the protocol was adapted for manual extraction using 2-mL tubes with a DynaMag™-2 Magnetic Rack (Thermo Fisher Scientific, Grand Island, NY; Cat. No. 12321D). The starting materials for extraction were individual wells on 96-well plates that contained a cell monolayer infected with virus and covered with 0.1 mL PBS. The plates were stored at -80°C at the appropriate timepoint (i.e., T0 or Tf) for RV-RT-PCR analysis. While still frozen, 0.1 mL RNA Lysis Buffer was added to each well to be extracted. Once thawed, the well contents with Lysis Buffer were pipetted up and down to mix prior to transferring to a RNase-free 2-mL snap-cap tube. The Magnesil protocol included a step with DNase I treatment, followed by additional wash steps, and final elution in 50 μL nuclease-free water with 0.5 μL RNasin® Plus RNase Inhibitor (Promega; Cat. No. N2611, 2500 units) or 0.125 μL RNasin Plus RNase Inhibitor (Promega; Cat. No. N2615, 10,000 units). If not analyzed immediately by RT-PCR, RNA extracts were stored at -80°C for up to 5 days.
6
Subcellular fractionation of HEK293T cells
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Soft lysis method was used for subcellular fractionation [74 (link)]. HEK293T cells were detached by treatment with 1× Trypsin, transferred into 1.5 mL tubes and centrifuged at room temperature, 168× g for 5′. The pellet was lysed with 175 µL/106 cells of cold RLN1 solution (50 mM Tris HCl pH 8, 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40, RNasin Plus RNase Inhibitor, Promega, Madison, WI, USA) and incubated 5′ on ice. Next, the suspension was centrifuged at 4 °C 300× g for 2′ and the supernatant, corresponding to the cytoplasmic fraction, was transferred into a new tube and stored on ice. The pellet containing nuclei was lysed with 175 µL/106 cells of cold RLN2 solution (50 mM Tris HCl pH 8, 500 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40, RNasin Plus RNase Inhibitor, Promega, Madison, WI, USA) and incubated on ice for 5 min. The suspension was centrifuged at 4 °C 16,360× g for 2′ and the supernatant, corresponding to the nuclear-soluble fraction, was transferred into a new tube and stored on ice. The remaining pellet corresponded to the chromatin-associated fraction. The ratio of target RNA in each fraction to total RNA was estimated using RT-qPCR. All experiments were performed in triplicate.
7
Grapevine Leaf Vein DNA and RNA Extraction
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Leaf veins (1 g) from asymptomatic and symptomatic field-grown grapevine plants used for insect feeding were minced on nitrogen liquid and DNA was extracted, according to Angelini et al. [96 ]. DNA was extracted from the S. titanus specimens one by one using the same protocol.
The total RNA was extracted from 50 mg of vegetable tissue from the micropropagated plantlets employed in the trial using the plant/fungi total RNA purification kit (Norgen), then it was added to 1 μl of the RNase inhibitor RNasin Plus (Promega) to avoid it degrading, and treated with DNAse (DNA-free kit Ambion).
The total RNA was extracted from 50 mg of vegetable tissue from the micropropagated plantlets employed in the trial using the plant/fungi total RNA purification kit (Norgen), then it was added to 1 μl of the RNase inhibitor RNasin Plus (Promega) to avoid it degrading, and treated with DNAse (DNA-free kit Ambion).
8
Nucleocytoplasmic Fractionation of A549 Cells
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Nucleocytoplasmic fractionation of A549 cells was performed as previously described80 (link). In short, 106 cells were scraped from 10 cm dish and washed with ice-cold PBS. The cells were resuspended in 0.2 mL of Dautry buffer (10 mM Tris-HCl pH 7.5, 140 mM NaCl, 1.5 mM MgCl2, 10 mM EDTA, 0.5 % NP-40, protease inhibitor cocktail cOmplete, Merck, RNase inhibitor RNasin Plus, Promega) and inclubated 3 min on ice before centrifugation for 5 min, 3.000 rpm at 4 °C. Supernatant (cytoplasmic fraction) was further cleared by centrifugation for 1 min at 15.000 g. Pellet (nuclei) was further washed twice with Dautry buffer and centrifugation for 5 min, 5.000 rpm at 4 °C, and finally lysed in SSB buffer (62.5 mM Tris HCl from 1 M stock solution with pH 6.8, 2% SDS, 10% Glycerol, 50 mM DTT and 0.01% Bromophenol Blue in distilled water) or Trizol (Thermo Fisher Scientific) for protein or RNA quantification, respectively.
9
Crosslinking of 293T cells
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1 × 106 293T cells were exposed to disuccinimidyl suberate (DSS; 1 mM, 0.3 mM, 0.1 mM), 1,8-bismaleimido-diethyleneglycol (BM(PEG)2; 1 mM, 0.3 mM, 0.1 mM) or N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS; 1 mM, 0.3 mM, 0.1 mM) crosslinking for 5 min at R.T. in PBS or permeabilised with 100 μL of reaction buffer including 125 mM NaCl, 50 mM Tris pH 7.0 (for BM(PEG)2) or 50 mM PIPES pH7.0 (for DSS or GMBS), 5 mM MgCl2 and 0.1% Triton X-100 at 37°C for 10 min before crosslinking. After quenching DSS with 20 mM Tris pH 7.0, BM(PEG)2 with 20 mM L-cysteine (Sigma-Aldrich) or GMBS with 20 mM Tris pH 7.0 and 20 mM L-cysteine, cells were processed for western blotting. 0.3 mM cross-linker was used for routine experiments while 1 mM was used for SAF-A IPs (Figures 4 C and 4E).
ForFigure 4 B right, reaction buffer was supplemented with 5 mM ATP or ATPγS. For Figure 4 D, after pre-treatment with 3 μg.ml-1 PureLink RNaseA (ThermoFisher) in reaction buffer at 37°C for 10 min, 293T cell lysates were incubated in the presence or absence of 30 μg.ml-1 total RNA from 293T cells and 5 units.ml-1 Apyrase (NEB) at 37°C for 10 min, following by crosslinking. After RNaseA treatment reaction buffer was supplemented with RNasin Plus RNase Inhibitor (Promega) 1000 units.ml-1, 1 mM DTT and 5 mM CaCl2.
For
10
In vitro Transcription and Xrn1 Treatment of 3'UTRs
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A DNA fragment corresponding to the RaTV 3′UTR under the T7 promoter was synthesized and cloned into the pIDT vector (IDT). Plasmids containing PCV 3′UTR and GFP gene fragments have been described previously [38 (link)]. For in vitro transcription, plasmids were first linearized by restriction digest and purified using a Monarch PCR and DNA Clean-up Kit (NEB). 3′UTRs were in vitro transcribed from 500 ng of plasmid DNA using a MEGAscript T7 Transcription Kit (Invitrogen). RNA was purified by LiCl precipitation and examined by electrophoresis in a 1 % denaturing agarose gel. RNA was then refolded in NEB3 buffer (85 °C for 5 min) and gradually cooled to 28 °C. For Xrn1 treatment, refolded RNA (1 µg) was incubated with 1 U Xrn1 (NEB) and 10 U RppH (NEB) in 20 µl of reaction mixture containing 1× NEB3 buffer (NEB) and 1 U µl–1 RNasin Plus RNase Inhibitor (Promega) for 2 h at 28 °C. The reaction was stopped by adding 20 µl of Loading Buffer II (Ambion), heating for 5 min at 85 °C and placing on ice. The denatured RNA samples were loaded into 6 % polyacrylamide TBE-Urea gels (Invitrogen), and electrophoresis was performed for 90 min in 1× TBE. The gels were stained with ethidium bromide and imaged using an OmniDoc imager (Cleaver Scientific).
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