RNA ladders in Figure 2B were generated by alkaline hydrolysis and RNase T1 digestion of 8 pmole fluorescently-labeled ssRNA target 25, followed by 3′ de-phosphorylation by T4 polynucleotide kinase (NEB). The reactions were then phenol: chloroform extracted and ethanol precipitated before 10% of each was analyzed by 15% UREA PAGE. Alkaline hydrolysis ladder was prepared by incubating the RNA target in 50 mM NaHCO3, pH 9.2, 1mM EDTA and 6.75 ng/μL yeast tRNA (Thermo Fisher Scientific) at 95°C for 20 minutes. RNase T1 ladder was prepared by combing the RNA target with 4U of RNase T1 (Thermo Fisher Scientific) in a 50 μL reaction (50 mM Tris, pH 7.5, 2 mM EDTA, and 3 mg/ml yeast tRNA) for 5 minutes at 37°C. The NmeCas9-catalyzed RNA cleavage reactions were treated by T4 polynucleotide kinase, phenol: chloroform extraction and ethanol precipitation, then analyzed alongside the RNA ladders on a 15% sequencing PAGE gel.
Yeast trna
Manufactured by Thermo Fisher Scientific
Sourced in United States
Yeast tRNA is a type of transfer ribonucleic acid (tRNA) isolated from the baker's yeast, Saccharomyces cerevisiae. tRNA molecules are responsible for the translation of genetic information from messenger RNA (mRNA) into proteins within cells. The core function of yeast tRNA is to facilitate the incorporation of amino acids into the growing polypeptide chain during protein synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Thermo Fisher Scientific (11 mentions)
Salmon sperm dna, by Thermo Fisher Scientific (8 mentions)
Bovine serum albumin (bsa), by New England Biolabs (7 mentions)
Dextran sulfate, by Merck Group (6 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Proteinase k, by Thermo Fisher Scientific (4 mentions)
Dynabeads myone streptavidin c1, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rnaseout, by Thermo Fisher Scientific (4 mentions)
Formamide, by Merck Group (4 mentions)
Ndei ncoi, by New England Biolabs (3 mentions)
T4 dna ligase, by New England Biolabs (3 mentions)
Proteinase inhibitor cocktail, by Roche (3 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Superase in rnase inhibitor, by Thermo Fisher Scientific (3 mentions)
Streptavidin magnetic beads, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (3 mentions)
Formamide, by Thermo Fisher Scientific (3 mentions)
149 protocols using yeast trna
1
RNA Ladder Generation and Analysis
2
Membrane Capture and Immunoprecipitation of Renatured Proteins
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DNA‐renatured samples were prepared as described above and aliquoted for immunoprecipitation or membrane capture. For membrane capture, approximately 40 μg renatured proteins or fragmented DNA alone (20 μg, negative control) were slowly passed through a 0.2 μm nitrocellulose membrane (1060004, GE Healthcare) pre‐blocked with 250 μg Yeast tRNA (Thermo Fisher Scientific). The membranes were then washed three times with 10 mM Tris–HCl pH 7.5, 100 mM KCl, 0.1% Triton X‐100; twice with 10 mM Tris–HCl pH 7.5, 500 mM KCl, 0.1% Triton X‐100; and once with 1X TE. Captured proteins and nucleic acids were eluted by the addition of 2% SDS/8M Urea and the nucleic acids extracted with Isol (5‐PRIME) as described above. Immunoprecipitation with Aβ 4G8 (1 μg) and mouse IgG (1 μg) were performed as described for RNA immunoprecipitation of renatured proteins above using approximately 40 μg of renatured proteins per IP. Protein A magnetic beads were pre‐blocked for 1 h at RT with 70 μg Yeast tRNA (Thermo Fisher Scientific). Sequencing libraries were prepared using the NEBNext DNA ultra kit (E7370, NEB), using NEBNext Indexing primers (E7335, NEB), and the libraries pooled and sequenced on the MiSeq system (Illumina) using paired‐end reads of 300 bp. Data can be accessed at GEO GSE99127.
3
In vitro RNase E Cleavage Assay
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RNase E in vitro cleavage assays were performed in 1× reaction buffer (25 mM Tris/HCl pH 7.5, 50 mM NaCl, 50 mM KCl, 10 mM MgCl2, 1 mM DTT) in a final volume of 10 μl as described earlier [31 ]. Briefly, radiolabelled GlmZ was mixed with 1 µg yeast tRNA (Ambion) and cold competitor RNA where appropriate. The RNA mix was heat-denatured, chilled on ice and incubated at 30°C for 5 min to allow for RNA folding. Afterwards, 20 nM RapZ was added and the samples were further incubated for 10 min at 30°C. Subsequently, 25 nM RNase E was added and incubation was continued for 30 min. The reactions were stopped by addition of 0.2 U proteinase K (NEB) and proteinase K buffer (100 mM Tris/HCl pH 7.5, 12.5 mM EDTA, 150 mM NaCl, 1% SDS) and incubation at 50°C for 30 min. Following the addition of 1 volume 2× RNA loading dye (95% formamide, 0.5 mM EDTA, 0.025% SDS, 0.025% bromophenol blue, 0.025% xylene cyanol) samples were separated on denaturing polyacrylamide gels (6% PAA, 7M urea, 1× TBE). Dried gels were analysed by phospho-imaging.
4
RNA-Protein Binding Assay
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RNA solution (0.2 μM) was prepared in 500 μl selection buffer supplemented with 0.5 M urea, and the RNAs were folded by heating to 80°C for 3 min followed by incubation at room temperature. The folded RNAs were mixed with 1 μl yeast tRNA (10 μg, Ambion) and 5 μl recombinant protein-immobilized beads, and incubated at 37°C for 30 min. After washing eight times with 500 μl sample buffer, RNAs were eluted with 50 μl 1× Buffer BXT Strep-Tactin XT Elution Buffer (IBA Lifesciences). The eluents were purified and concentrated by ethanol precipitation and then dissolved in 10 μl TE.
5
La-Module Binding Kinetics with RNA
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RNA oligonucleotides were 5ʹ-end labelled with [γ-32P]-ATP (Perkin Elmer) using T4-polynucleotide kinase (New England Biolabs) and gel purified. Capped RPS6 20-mer was prepared as previously described[25 (link)]. La-Module 5X stocks were prepared in dilution buffer (50 mM Tris-HCl pH, 7.5, 100 mM NaCl, 25% glycerol, 4 mM DTT). 10 μL binding reactions contained final concentrations of 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 7.5% v/v glycerol, 1mM DTT, 1 μM BSA (Thermo Fisher Scientific), ≤ 2 nM radiolabeled RNA, and either: 10 U/mL poly(dI-dC) (Sigma-Aldrich), 5 μM Yeast tRNA (Ambion) or 3 μM salmon sperm DNA (Thermo Fisher Scientific). La-Module was titrated at 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 μM. Reactions were incubated on ice for 30 min then run on 7% polyacrylamide (29:1) native 0.5X TBE gels at 125 V for 45 min at 4°C. Exposed phosphor screens (GE Healthcare Lifesciences) were imaged on a Typhoon FLA plate reader (GE Healthcare Lifesciences) and quantitated using Imagequant TL (GE Healthcare Lifesciences). Dissociation constants were determined by plotting (KaleidaGraph) the fraction of shifted RNA versus the concentration of protein after band intensities were corrected for background (ImageQuant)TL.
6
Thermal Shift Assay for RNA Binding
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To assay RNA binding, a dilution series of 25 nt model RNAs, up to 16 μm , were mixed with 2 μm protein and 1:500 protein thermal shift dye in 20 mm HEPES-NaOH, pH 7.5, 150 mm KCl, 5% glycerol, 1 mm DTT, and 20 ng/μl yeast tRNA (Ambion). For testing the stability of IFIT proteins and complexes, in an optical 96-well reaction plate (Applied Biosystems), 2.5 µg of protein was mixed with 1:500 protein thermal shift dye (Life Technologies) in 20 mm HEPES-NaOH, pH 7.5, 150 mm KCl, 2.5 mm MgOAc, 5% glycerol, and 1 mm DTT, in a final volume of 20 μl. Emission was measured at 623 nm in a ViiA7 real-time PCR system (Applied Biosystems), ramping from 25 to 95 ˚C stepwise at a rate of 1 ˚C per 20S. For interpolation of melting temperatures, the data were analyzed using the Bolzmann equation (y = LL + (UL –LL)/(1 + exp(Tm – x)/1)), where LL and UL are the minimum and maximum fluorescence intensities, respectively, and melting temperature (Tm) was interpolated from the 50% intersect of the curve.
7
ChIA-PET Library Preparation Protocol
8
ChIA-PET Library Preparation Protocol
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ChIA-PET library was prepared as previously described28 (link) with the following modifications. The proximity ligated chromatin complex was eluted with 1% SDS (Ambion) and de-crosslinked using proteinase K (Invitrogen) and purified using Zymo ChIP DNA Clean & Concentrator (Zymo Research). The purified fragments were tagmented using the Nextera DNA Sample Preparation Kit (Illumina). The linker ligated ChIA-PET constructs were selected using Sera-Mag Speed Beads Streptavidin-Blocked Magnetic Particles (GE Healthcare). The magnetic beads were blocked with yeast tRNA (Ambion) prior to the selection. The streptavidin selected constructs were amplified with 8–10 cycles of PCR and purified using Ampure XP beads (Beckman Coulter). For the primary mES B6NJ and KO cell clones, the library were prepared with an in situ approach where crosslinked cells were lysed and digested with AluI enzyme (NEB). Fragmented DNA ends were A-tailed and ligated with biotinylated linker overnight58 . The ligated chromatin were then sheared by sonication and immunoprecipitated with anti-Ezh2 antibody. The immunoprecipitated DNA were subjected to tagmentation, biotin selection and amplication. Libraries were sequenced on Illumina Nextseq, Hiseq and Novaseq platforms.
9
Biotinylated miRNA-214-3p Pulldown Assay
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Biotinylated (Dharmacon, Lafayette, CO, USA) pull-down assay was performed as described earlier [12 (link)]. Briefly, 1 × 106 TE7 cells were transfected with biotin-labelled miR-214-3p or control miR at a final concentration of 50 nM for 48 h. Whole cell lysates were incubated at 4 °C overnight with 50 μl/sample of streptavidin-Dyna beads (Invitrogen, Carlsbad, CA, USA) coated with yeast tRNA (Ambion, Austin, TX, USA). Next day, beads were washed thoroughly and RNA was isolated using TRIzol (Invitrogen) and standard chloroform-isopropanol method and then subjected to PCR as explained above.
10
RNA-Protein Binding Assay with KhpA and KhpB
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Labeled RNAs and yeast tRNA (Ambion, 1 mg/ml) were denatured by heating for 1 min at 95°C and cooling on ice for 1 min. Four nanomolar of each labeled RNA and 1 μg of yeast tRNA were incubated with increasing concentrations (0, 0.16, 0.32, 0.63, 1.25, 2.5, 5 μM) of KhpA, KhpB-ΔN or both proteins (in a 1:1 ratio) in binding buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM MgCl2) at 37°C for 1h. The reactions were stopped by adding 5× RNA native loading buffer (0.5× TBE, 50% glycerol, 0.2% xylene cyanol, 0.2% bromophenol blue). The samples were loaded on a pre-cooled native 6% polyacrylamide gel and separated by running the gel at 4°C in 0.5% Tris–borate–EDTA (TBE) buffer at a constant current of 40 mA for 4 h. The gel was then vacuum-dried, and signals were detected on a Typhoon FLA 7000 phosphoimager (GE Healthcare).
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