Biotin-labeled RNA was synthesized by in vitro transcription in the total reaction volume of 20 μl at 37 °C for 3 h with 1 μg pCDNA3-HMS/HOXC10 3′UTR using RiboMAX Large Scale RNA Production Systems (P1300) from Promega biotech as per the manufacture instructions along with 2 μM biotin-14-CTP (Invitrogen). Loading solution (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol) was added to the reaction products and analyzed on an 8% denaturing agarose gel. For RNA pull-down assay, the cell lysate was incubated with biotin-labeled RNA and streptavidin magnetic beads followed by washing, elution, and immunoblotting to detect the RNA-associated proteins. For detecting associated lncRNAs, the RNA was extracted from the eluate using TRIzol and reverse transcribed into cDNA using ThermoScript RT-PCR System kit from Invitrogen as per the manufacture instructions. The qRT-PCR reactions were carried with specific primers to identify associated RNA.
Biotin 14 ctp
Manufactured by Thermo Fisher Scientific
Sourced in United States
Biotin-14-CTP is a nucleotide analog that incorporates biotin into RNA transcripts. It can be used for various molecular biology applications such as RNA labeling and detection.
Automatically generated - may contain errors
Lab products found in correlation
Microspin g 25 column, by GE Healthcare (2 mentions)
Rq1 dnase, by Promega (2 mentions)
Ribomax large scale rna production system t7, by Promega (2 mentions)
Maxiscript t7 transcription kit, by Thermo Fisher Scientific (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Imagequant tl, by GE Healthcare (2 mentions)
Typhoon phosphorimager, by GE Healthcare (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Thermoscript rt pcr system kit, by Thermo Fisher Scientific (1 mentions)
Ribomax large scale rna production system, by Promega (1 mentions)
Streptavidin beads, by Merck Group (1 mentions)
In vitro transcription system, by Promega (1 mentions)
T7 rna polymerase, by Promega (1 mentions)
Megascript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Streptavidin sepharose, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Active Motif (1 mentions)
Riboprobe in vitro transcription system, by Promega (1 mentions)
Megascript kit, by Thermo Fisher Scientific (1 mentions)
Dna polymerase 1 klenow, by New England Biolabs (1 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (1 mentions)
13 protocols using biotin 14 ctp
1
Biotin-labeled RNA Synthesis and Pulldown
2
RNA-Protein Interaction Pulldown Assay
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The biotin-labeled RNA probe was synthesized by in vitro transcription system (Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, Aurora-A 5′-UTR was transcribed by T7 RNA polymerase (Promega) and incorporated with biotin-14-CTP (Invitrogen, Carlsbad, CA, USA) at 37 °C for 1.5 h. After treatment with RQ1 DNase (Promega), the biotin-labeled RNA probe was purified using the illustra MicroSpin G-25 Columns (GE Healthcare, Chicago, IL, USA). Total cell lysates collected by RIPA buffer were pre-cleaned by streptavidin beads (Sigma, St. Louis, MO, USA) at 4 °C for 1 h. For pull-down assay, the total cell lysates were diluted with 5X EMSA buffer (50 mM Hepes [pH 8], 250 mM KCl, 10 mM MgCl2, 5 mM DTT and 25% glycerol) including 0.5 mg/ml tRNA, 0.7 mg/ml heparin, 100 unit/ml RNase inhibitor, protease inhibitors and biotin-labeled RNA probes. After incubating at 4 °C for 2 h, the mixtures were subjected to UV-crosslinking (120 mJ/cm2) for 5 min three times, and then streptavidin beads were added to rotate at 4 °C for 1 h. The beads were washed by the wash buffer (10 mM Hepes (pH 8), 40 mM KCl, 3 mM MgCl2, 2 mM DTT, 5% glycerol, 0.5% SDS and 2% NP-40) three times, followed by western blot analysis.
3
Biotinylated circRNA Synthesis Protocol
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As DNA templates for in vitro transcription, pcDNA3/circRNA vectors encoding the full-length exonic sequences of five human circRNAs were used. Biotinylated RNAs were produced from 0.5 µg linearized, and phenol/chloroform extracted template using the MEGAscript T7 Transcription Kit (Ambion, Austin, Texas, United States), according to the manufacturer’s protocol with addition of 0.75 mM Biotin-14-CTP (Invitrogen) to the transcription reaction. In controls, nuclease free water was added instead of Biotin-14-CTP. The transcribed RNA was purified by phenol/chloroform extraction and dissolved in nuclease free water.
4
Biotinylated RNA Affinity Purification
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We synthesized 20-nucleotide biotinylated RNA probes with the T7 RiboMAX large-scale RNA production system (Promega) using 3.0 mM Biotin-14-CTP (Invitrogen) as described previously38 (link). The template DNA was generated as described for RNA-EMSA.
The RNA affinity purification method was modified from the previously adopted protocol38 (link). Biotinylated RNAs (0.75 nmol) and HeLa nuclear extract (30 μl) (CilBiotech) were mixed in a 500-μl binding buffer [20 mM HEPES, pH 7.8, 150 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.05% Triton X, 1× Protease Inhibitor Cocktail (Active Motif)], and were incubated at 30°C for 3 h with gentle agitation. In parallel, 50 μl streptavidin-conjugated beads (Streptavidin-sepharose, GE Healthcare) were blocked with a 1:1 mixture of 1 ml binding buffer containing yeast tRNA (0.1 mg/100 μl of beads) and 1 ml PBS containing 4% BSA at 4°C with rotation for 1 h. The beads were mixed with the binding solution for 2 h at 4°C with gentle rotation. After washing the beads four times with 1 ml binding buffer, RNA-bound proteins were eluted in SDS loading buffer by boiling at 95°C for 5 min. The isolated proteins were fractionated on a 10% SDS-polyacrylamide gel and stained with Coomassie blue or by immunoblotting.
The RNA affinity purification method was modified from the previously adopted protocol38 (link). Biotinylated RNAs (0.75 nmol) and HeLa nuclear extract (30 μl) (CilBiotech) were mixed in a 500-μl binding buffer [20 mM HEPES, pH 7.8, 150 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.05% Triton X, 1× Protease Inhibitor Cocktail (Active Motif)], and were incubated at 30°C for 3 h with gentle agitation. In parallel, 50 μl streptavidin-conjugated beads (Streptavidin-sepharose, GE Healthcare) were blocked with a 1:1 mixture of 1 ml binding buffer containing yeast tRNA (0.1 mg/100 μl of beads) and 1 ml PBS containing 4% BSA at 4°C with rotation for 1 h. The beads were mixed with the binding solution for 2 h at 4°C with gentle rotation. After washing the beads four times with 1 ml binding buffer, RNA-bound proteins were eluted in SDS loading buffer by boiling at 95°C for 5 min. The isolated proteins were fractionated on a 10% SDS-polyacrylamide gel and stained with Coomassie blue or by immunoblotting.
5
Biotin-labeled 5' UTR Synthesis
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To synthesize the biotin-labeled 5′ UTR, the plasmids including K1-pGEM-T-Easy, K2-pGEM-T-Easy and nucelolin-pGEM-T-easy were linearized through restriction enzyme digestion using NdeI. Three biotin-labeled 5′ UTR were synthesized using the Riboprobe in vitro Transcription System (Promega). Following manufacturer's instructions, 1 μg of linearized DNA was incubated with the transcription optimized buffer (100 mM DTT, 2.5 mM γATP, 2.5 mM γGTP, 2.5 mM γUTP, 100 μM γCTP), 10 mM biotin-14 CTP (Invitrogen), 1 unit/μl of recombinant RNase ribonuclease inhibitor and T7 RNA polymerase for 2 h at 37°C. The reaction mixture was then treated with RQ1 DNase (Promega) for 15 min at 37°C and purified using Microspin G-25 columns (GE Healthcare).
6
Promoter Sequences of HEV Genotype 1
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Sequences coding for the putative genomic promoter (G promoter: nt 1 to 139 on positive sense RNA) and putative sub-genomic promoter (Sg promoter: nt 5051 to 5200 on positive sense RNA) of HEV genotype 1 were PCR amplified from pSK-HEV2 replicon. The primers for the amplification of template were designed with T7 promoter sequence in such a way that the RNA of anti-sense orientation is generated. Primers used for the amplification have been listed in Table 1 . PCR products were used as templates for the synthesis of RNAs bearing respective promoter sequences. In vitro RNA was synthesized by using MEGAscript kit (Ambion) following the manufacturer’s instructions. Biotinylated in vitro transcribed RNAs were prepared using 5 mM rATP, 5 mM rGTP, 5 mM rUTP, 4.5 mM rCTP, and 0.5 mM of biotin-14 CTP (Invitrogen) in the rNTP mix for the in vitro transcription reaction. For synthesizing non-biotinylated RNAs of respective regions, total 5 mM rCTP was added instead of biotin-14-CTP. Unincorporated nucleotides were removed by purifying the RNA using phenol-chloroform precipitation method. Purified RNAs were visualized on 2% agarose gel.
7
Characterization of G4 RNA Structures
8
Biotin-Labeling of DNA Fragments
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The overhangs generated by the restriction enzyme cuts were filled-in by adding a mix of biotin-14-CTP (0.4 mM stock; 18.75 μL for the 5 M to 100 k samples or 10 μL for the 50 k to 1 k samples; Life Technologies), 10 mM dATP (whichever was not supplied in biotinylated form), dGTP and dTTP (10 mM stocks; 0.75 μL of each dinucleotide for the 5 M to 100 k samples or 0.5 μL for the 50 k to 1 k samples), and 5 U/μL DNA polymerase I Klenow (5 M to 100 k samples or 4 μL for the 50 k to 1 k samples; New England Biolabs), followed by a 90 min incubation at 37 ° C with gentle rotation (20 rpm).
9
Biotinylated SNCA 3'UTR RNA Synthesis
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The biotinylated RNA probes of SNCA short 3′UTR (3′UTRS) and long (3′UTRL) were synthesized by in vitro transcription from linearized pBSK plasmid vector by using a T7 MegaScript kit (Ambion) with addition of biotin-14-CTP (Life Technologies). RNA concentration and integrity were verified by NanoDrop 1000 spectrophotometer (Thermo Scientific) and by denaturing agarose gel electrophoresis of the RNA. Successful biotinylation was checked using the Chemiluminescent Nucleic Acid Detection Module kit (Thermo Scientific).
RNA affinity purification was performed as described by Hammerle M. with some adaptations (10 ) (seeSupplementary Materials for details).
RNA affinity purification was performed as described by Hammerle M. with some adaptations (10 ) (see
10
Characterization of G4 RNA Structures
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G4-forming PIM1 and control ΔG4 sequences were taken from 48 (link). Two additional control RNAs, one for which the Gs within the G4-forming sequence were mutated to non-Gs (G-to-H) and a second for which the Gs within the G4-forming sequence were mutated to non-Gs and an equal number of non-G nucleotides outside of the G4-forming sequence were mutated to Gs (G-rich), were synthesized as gBlocks (IDT, sequences in Supplementary Table 2 ) and cloned into pcDNA3.1. Linearized vectors were transcribed using the MAXIscript T7 Transcription Kit (Thermo Fisher Scientific) and RNA treated with Turbo DNase (Thermo Fisher Scientific). Biotin-14-CTP (19519016 Life Technologies) was added in a 0.4:1 ratio relative to CTP. RNA integrity was verified by polyacrylamide gel electrophoresis. G4 structure formation was confirmed using a reverse transcriptase stalling assay 68 (link). [rG4rA4]5, 5’-biotinylated-[rG4rA4]5, [rGrA]20 and 5’-biotinylated-[rGrA]20 40-mer RNA oligonucleotides were obtained from IDT. Native gel electrophoresis to measure formation of secondary structure was performed as described 35 (link). RNA was folded either as described 35 (link) or in pull-down buffer to confirm maintenance of RNA structure during PRC2 pull-down assays. Radiolabeled RNA was visualised using a Typhoon phosphorimager (GE) and ImageQuantTL (GE).
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