The aminoacylation assay was carried out in buffer containing 4 mM DTT, 50 mM HEPES-KOH pH 7.6, 20 mM KCl, 10 mM MgCl2, 5 mM ATP, 2 mg mL-1 yeast tRNA (Roche), various concentrations of [3H] Thr (American Radiolabeled Chemicals), and 100 nM TRS. Reactions were initiated with enzyme and conducted in a 37 °C heat block. Aliquots (10 μL) were taken at different time points and quenched on Whatman filter pads presoaked with 5% trichloroacetic acid (TCA). Pads were washed three times for 10 min each time with cold 5% TCA and once with cold 100% ethanol, and dried, and radioactivity was quantified using a scintillation counter (Beckman Coulter).
Yeast trna
Manufactured by Roche
Sourced in United States
Yeast tRNA is a type of transfer ribonucleic acid (tRNA) isolated from the yeast Saccharomyces cerevisiae. tRNA molecules play a crucial role in protein synthesis by facilitating the translation of genetic information from messenger RNA (mRNA) into polypeptide chains. Yeast tRNA serves as a standard reagent for various molecular biology applications, such as in vitro translation systems and tRNA structure studies.
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Lab products found in correlation
Scintillation counter, by Beckman Coulter (2 mentions)
Whatman filter pads, by Cytiva (2 mentions)
Cfx384 instrument, by Bio-Rad (2 mentions)
Hard shell 384 well microplate, by Bio-Rad (2 mentions)
Microseal b clear, by Bio-Rad (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Freedom evo 150, by Tecan (2 mentions)
Typhoon imager, by GE Healthcare (1 mentions)
6 fam, by Merck Group (1 mentions)
Ap conjugated anti dig antibody, by Roche (1 mentions)
Horseradish peroxidase hrp conjugated anti dig antibody, by Roche (1 mentions)
Immpact dab substrate, by Vector Laboratories (1 mentions)
4 nitroblue tetrazolium chloride, by Roche (1 mentions)
Heparin, by Merck Group (1 mentions)
Streptavidin magnesphere paramagnetic particles, by Promega (1 mentions)
Rnasin, by Promega (1 mentions)
Heparin, by Nacalai Tesque (1 mentions)
Nitroblue tetrazolium 5 bromo 4 chloro 3 indolyl phosphate, by Roche (1 mentions)
B m blocking reagent, by Roche (1 mentions)
Proteinase k, by Roche (1 mentions)
11 protocols using yeast trna
1
Aminoacylation Assay for Threonine
2
Purification of Hexanucleotide Repeat-Binding Proteins
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Purification of hexanucleotide repeat-binding proteins was performed following our previously described protocol [41 (link)]. In brief, a total of 0.6 mg of HeLa cell nuclear extract was diluted in 4 ml of protein-binding buffer (10% glycerol, 10 mM HEPES, 50 mM KCl, 1 U/ml RNase inhibitor, 0.15 lg/ml yeast tRNA (10109495001, Roche), 1 mM EDTA, 1 mM DTT and 0.5% Triton X100 in DEPC water). The diluted extract was then precleared with heparin-agarose (H6508, Sigma-Aldrich) and streptavidin-agarose (15942-050, Invitrogen). The precleared nuclear extracts were incubated with streptavidin μMACS-microbeads (MACS molecular) and 150 pmol of biotinylated repeat RNA in the presence of 50 mM KCl for 1 h. For competition experiments, nuclear extracts were incubated for 1 h with 7.5 nmol (50-fold excess) of non-biotinylated (C4G2)17 competitor RNA before addition of the biotinylated probe. The reaction mixture was loaded on a μMACS column and subsequently washed three times with protein-binding buffer. Hexanucleotide repeat binding proteins were sequentially eluted with increasing concentrations of NaCl. Each eluate was TCA precipitated and subjected to SDS-PAGE.
3
Dilution Series for qPCR Optimization
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This data set created a dilution series consisting of four 10-fold serial dilution points from 15,000 to 15 molecules, using 10 ng / μl yeast tRNA as a carrier (Roche) and created NTC samples of the same dilution. qPCR was done on a CFX 384 instrument (Bio-Rad). QPCR was performed on a CFX 384 instrument (Bio-Rad) using a 96-well pipetting robot (Tecan Freedom Evo 150). Amplification reactions were performed in 8 μl samples containing 0.4 μl forward and 0.4 μl reverse primer (5 μM each), 0.2 μl nuclease-free water, 4 μl iQ SYBR Green Supermix (Bio-Rad) and 3 μl of standard oligonucleotide. In 384-well plates (Hard-Shell 384-well microplate and Microseal B clear using an adhesive seal (Bio-Rad)), for each of the 4 dilution points, a total of 94 replicate reactions were distributed. In addition, the NTC reaction was repeated 8 times [28 (link)]. This dataset will be referred to as ‘94-replicates-4-dilutions set’. And 44 (4 × 11) amplification curves of the MYCN gene with a diluted concentration of 15, 150, 1500,15,000(11 replicated experiments for each group) were used for subsequent analysis.
4
Fluorescent Electrophoretic Mobility Shift Assays
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EMSAs for the SAP domain were performed with DNA such that the single strand or one of the two strands in dsDNA was 5′-labeled with fluorescein (6-FAM, Sigma-Aldrich) (Table 1 ). For each reaction, 0.1 pmol of either single- or double-stranded DNA were mixed with 0,6 µg of yeast tRNA (Roche), 10 mM MgCl2 and SAP-buffer (25 mM sodium phosphate, 150 mM NaCl, 0.02% NaN3, pH 6.5)/protein in different volumes/concentrations to match a final reaction volume of 20 µL. Additionally, 3 µL of loading buffer were added immediately prior to loading the samples (10 µL of each reaction) onto a 6% polyacrylamide gel. Gel-electrophoresis was run for 50 min at 80 V. Finally, the gels were imaged inside the glass plates with a Typhoon Imager (GE Healthcare) with a laser at 488 nm excitation and an emission filter at 520 nm.
5
Quantitative Aminoacylation Assay Protocol
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Aminoacylation assays were performed as previously described.21 (link) Briefly, assays were carried out in a buffer containing 4 mM DTT, 50 mM HEPES-KOH (pH 7.6), 20 mM KCl, 10 mM MgCl2, 5 mM ATP, 2 mg ml−1 yeast tRNA (Roche, Indianapolis, IN, USA), threonine, L-[3-3H] (American Radiolabeled Chemicals), and cell lysates. Reactions were initiated with cell lysates and conducted at 37 °C. Aliquots (20 μl) were taken from the reactants after 30 min and quenched on Whatman filter pads that were presoaked with 5% TCA. Pads were washed three times for 10 min each time with cold 5% TCA, and once with cold 100% ethanol. Washed pads were then dried, and radioactivity was quantified using a scintillation counter (Beckman Coulter, Brea, CA, USA).
6
In situ Hybridization with Labeled RNA Probes
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DIG-labeled cRNA probes were diluted to 500 ng/mL in hybridization solution; 50% formamide, 2× saline-sodium citrate buffer, 1× Denhardt’s solution, 100 µg/mL salmon sperm DNA, and 300 µg/mL yeast tRNA (Roche Diagnostics). Mouse sections were hybridized with cRNA in a hybridization buffer overnight at 65 °C (human sections; 60 °C). The sense probe was used as a negative control. After hybridization, sections were incubated with alkaline phosphatase (AP)-conjugated anti-DIG antibody (Roche Diagnostics) or horseradish peroxidase (HRP)-conjugated anti-DIG antibody (Roche Diagnostics). Labeled RNA was detected with 4-nitroblue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indoyl-phosphate (BCIP) mixture (Roche Diagnostics) for AP and the ImmPACT DAB substrate (Vector Lab., Burlingame, CA, USA) for HRP, respectively.
7
RNA-Protein Binding Assay with ZAP-S
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Different amounts (0, 1, 5, and 10 pmole) of biotin-labeled RNA probe were incubated with 100 μg of ZAP-S-V5 or ZAP-S-del4ZFs expressing A549 cell extracts lysed by CHAPS lysis buffer (10 mM Tris-HCl [pH 7.4], 1 mM MgCl2, 1 mM EGTA, 0.5% CHAPS, 10% glycerol, and 5 mM 2-mercaptoethanol) containing protease inhibitor cocktail in a final volume of 100 μl of RNA binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM EDTA, and 10 μM ZnCl2) supplemented with 1 unit/μl RNasin (Promega), 1 μg/μl heparin (Sigma-Aldrich) and 100 ng/μl yeast tRNA (Roche) for 30 min at 30°C. After incubation, 300 μl of Streptavidin MagneSphere paramagnetic particles (Promega) was added for 30 min at 25°C. Protein-RNA complexes were washed three times by RNA binding buffer without heparin and yeast tRNA. After washing, 30 μl of 2X SDS-PAGE sample buffer was added and incubated for 10 min at room temperature. The pull-down proteins were further analyzed by western blot.
8
In Situ Hybridization of Mouse Embryos
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Mouse embryos were obtained after euthanasia by cervical dislocation (E14.5 of Mkx+/− mice). The samples were fixed overnight in 4% PFA/PBS and processed for frozen sectioning (16 μm). Sections were treated with 10 μg ml−1 proteinase K (Roche) for 10 min at room temperature (RT) and then acetylated with acetylation solution for 20 min at RT. Pre-hybridization (in × 5 SSC (pH 4.5), 50% formamide, 1% SDS, 50 μg ml−1 yeast tRNA (Roche) and 50 μg ml−1 heparin (Nacalai Tesque)) was performed at 68 °C for 2 h; then, a DIG–RNA probe (500 ng ml−1) was added and hybridized for 14 h at 68 °C. Subsequently, sections were subjected to a series of post-hybridization washes in wash buffers containing formamide, SSC, SDS and 0.05% CHAPS. After blocking with 2% BM Blocking reagent (Roche) containing 0.1% Tween 20 (TBST) for 30 min at RT, embryos were incubated with anti-DIG-AP Fab antibody fragments (Roche) and 1% sheep serum in TBST for 3 h at RT. After a series of washes with TBST, embryos were equilibrated with NTMT (5 M NaCl, 1 M Tris–HCl (pH 9.5), 1 M MgCl2 and 0.1% Tween 20). Colour development reactions were performed at 4 °C or RT with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche).
9
Cell Culture and Aptamer Modification
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MHCC97L cell and HCCLM9 cell were obtained from research center of Zhongnan Hospital, Wuhan University, as we previously described [29 (link)] and cultured in RPMI1640 (Gibco) containing 10% FBS (Gibco) and 100 units/mL penicillin-streptomycin (Beyotime, Shanghai, China). Other cell lines were maintained at our laboratory. Salmon sperm DNA, yeast tRNA, and BSA were purchased from Roche (F. Hoffmann-La Roche Ltd., USA). Streptavidin-coated magnetic nanoparticles M-280 (Dynabeads) were used for modifying biotin-labelled single-stranded DNA (ssDNA) aptamers pool.
10
Comprehensive RNA Isolation and Library Preparation
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Yeast-tRNA (Roche 10109525001), RNase inhibitor (Promega N251A), RNaseZap (Invitrogen AM9780), TRIzol Reagent (Invitrogen 15596018), 3 M Sodium Acetate pH5.5 (Invitrogen AM9740), Perfect Start Green qPCR SuperMix (TransGene AQ601-04), RNeasy RNA purification Kit (Qiagen 74104), mRNA Capture Beads (Vazyme N401), Qubit RNA Assay Kit (Qubit Q32852), RNA secure Reagent (Ambion AM7006), Turbo DNase (Invitrogen AM2238), riboPOOLs (rRNA removal probes) (siTOOLs Biotech), Streptavidin magpoly beads (SMART lifesciences SM01710), Low Melting Point Agarose (Invitrogen 16520-050), 10 × TBE Buffer (Invitrogen 15581-044), DNA size Marker (DL1000, Takara 3591Q), NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760).
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