The RNA pull-down assay was performed according to instructions (Pierce Magnetic RNA-Protein Pull-Down Kit, 20164). In brief, artificially synthesized tRF-Gln-CTG-026 was biotinylated. Streptavidin Magnetic Beads (NEB, S1420S) were pre-washed, and the cell lysate was prepared; the protein concentration was required to be more than 2 mg/mL. The Streptavidin Magnetic Beads were washed with 50 μL 20 mM Tris (v/v), and 50 μL 1 × RNA Capture Buffer was added and mixed well. After 50 pM labeled RNA was added, the solution was kept at room temperature for 1 h. Then, 20 mM Tris (pH 7.5) (v/v) was added to wash the beads. Protein–RNA Binding Buffer (1×) was added into the beads and mixed well. RNA–protein mix was added to the beads for 3 h at 4 °C. Finally, the samples were washed and eluted.
Streptavidin magnetic beads
Manufactured by New England Biolabs
Sourced in United States
Streptavidin magnetic beads are a laboratory product consisting of magnetic particles coated with the protein streptavidin. Streptavidin has a high affinity for the small molecule biotin, allowing the beads to be used for various biotechnology applications that involve the biotin-streptavidin interaction.
Automatically generated - may contain errors
Lab products found in correlation
T7 rna polymerase, by Roche (4 mentions)
Biotin rna labeling mix, by Roche (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Biotin 11 ddutp, by Thermo Fisher Scientific (2 mentions)
Nebuffer 3, by New England Biolabs (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Sodium borate, by Thermo Fisher Scientific (2 mentions)
Hrp linked anti rabbit igg, by Cell Signaling Technology (2 mentions)
Hrp linked anti mouse igg, by Cell Signaling Technology (2 mentions)
Ring1b, by Cell Signaling Technology (2 mentions)
Eb buffer, by Qiagen (2 mentions)
Nu 1619 biox s, by Jena Biosciences (2 mentions)
Nu 809 biox s, by New England Biolabs (2 mentions)
Spriselect, by Beckman Coulter (2 mentions)
T4 pnk, by New England Biolabs (2 mentions)
Glycanassure apts kit, by Thermo Fisher Scientific (2 mentions)
3500xl genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Rabbit reticulocyte lysate, by Promega (2 mentions)
Annealed dna ultramers, by Integrated DNA Technologies (2 mentions)
Biotin 11 utp, by PerkinElmer (2 mentions)
84 protocols using streptavidin magnetic beads
1
RNA-Protein Pull-Down Assay Protocol
2
Magnetic Bead-based DNA Nicking Assay
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Streptavidin-magnetic beads (1 μm diameter, 4 mg mL−1, an aqueous suspension containing 0.1% bovine serum albumin, pH 7.4, 0.05% Tween-20, and 0.05% NaN3), nicking endonuclease (Nb.BbvCI) and 10× CutSmart™ buffer (20 mM, Tris-acetate, 500 mM potassium acetate, 10 mM magnesium acetate, and 100 μg mL−1 BSA, pH 7.9) were purchased from New England Biolabs (Beijing, China). Gold View I (GV I) (10 000×) used in this study was provided by Solarbio Co. Ltd. (Xiamen, China). The DL 200 DNA marker was ordered from Takara (Dalian, China). Salmon sperm DNA was obtained from Solarbio Inc. (Beijing, China). All oligonucleotides were synthesized and purified via HPLC by Sangon Biotechnology Co. Ltd. (Shanghai, China), and the corresponding sequences are illustrated in Table S1 (ESI).† All oligonucleotides were dissolved in tris-ethylenediaminetetraacetic acid (TE) buffer (pH 8.0, 10 mM Tris–HCl, 1 mM EDTA) and stored at −20 °C for further use.
Moreover, 0.1 M phosphate-buffered solution (PBS) included 136.89 mM NaCl, 2.67 mM KCl, 8.10 mM Na2HPO4 and 1.76 mM KH2PO4 (pH 7.4). The reaction buffer contained 10 mM Tris–HCl, 1 mM EDTA and 12.5 mM MgCl2 (pH 8.0). Other chemicals (analytical grade) were obtained from standard reagent suppliers. Millipore-Q water (≥18 MΩ, Milli-Q, Millipore) was used throughout the experiments.
Moreover, 0.1 M phosphate-buffered solution (PBS) included 136.89 mM NaCl, 2.67 mM KCl, 8.10 mM Na2HPO4 and 1.76 mM KH2PO4 (pH 7.4). The reaction buffer contained 10 mM Tris–HCl, 1 mM EDTA and 12.5 mM MgCl2 (pH 8.0). Other chemicals (analytical grade) were obtained from standard reagent suppliers. Millipore-Q water (≥18 MΩ, Milli-Q, Millipore) was used throughout the experiments.
3
Nascent chromatin capture assay protocol
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The nascent chromatin capture assay was performed as described previously (48 (link)) with slight modifications. Cells were treated with a mixture of biotin-16-dUTP and biotin-16-dCTP (0.5 μM each, Jena Bioscience) in hypotonic buffer (50 mM KCl, 10 mM HEPES) for 5 min and followed by another 5 min dUTP/dCTP treatment in regular DMEM medium (For etoposide treated samples, etoposide was added 20 min prior dUTP/dCTP treatment and kept until cells were fixed). Cells were then fixed by 0.2% PFA for 5 min at room temperature and quenched by co-incubation with 200 mM glycine for 1 min. Cells were resuspended in TNT-300 buffer (25 mM Tris–HCl pH 7.4, 300 mM NaCl, 1% Triton-X100) together with protease and phosphatase inhibitors (Sigma, 1:1000) and sonified (30% amplitude, 45 s on/15 s off for 10 min) before incubated with 10 μl of Streptavidin Magnetic Beads (50% slurry, NEB) at room temperature 45 min on a rotating wheel. Beads were collected by centrifugation and washed three times with the TNT-300 buffer. Samples were denatured by incubation with the 4× Laemmli loading buffer at 95°C for 10 min and analyzed by immunoblotting.
4
Biotin-labeled DNA Pulldown Assay
5
Nanoparticle-Based EXPAR Assay
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EXPAR templates, DNA targets and DNA probe functionalized on AuNPs were purchased from Integrated DNA Technologies. The sequences of DNA used are shown in Table 2 . The Vent (exo-) polymerase, Nt.BtsNBI nicking enzyme, the ThermoPol buffer, the NEBuffer 3.1, BSA, SSB proteins, dNTPs and the Streptavidin magnetic beads, were purchased from New England BioLabs. The Biotin-11-dUTP was obtained from Biotium. Ethylene glycol, propylene glycol, betaine, DMSO, trehalose, TMAC, and HAuCl4, sodium citrate dehydrate, 4-mercaptobenzoic acid (MBA) were purchased from Sigma-Aldrich.
6
CRISPR DNA Pulldown Assay
7
Magnetic Bead-Based Protein Enrichment
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A 20 μl aliquot of streptavidin magnetic beads (New England Biolabs) was washed with once with 200 μl Buffer A (10 mM Tris pH 8.0, 50 mM NaCl, 10 mM CaCl2, 0.01% Tween 20) and resuspended in 50 μl of Buffer A containing 500 ng of biotinylated-His-HpaII. After pipette mixing to allow the His-HpaII to bind to the beads, the His-HpaII-beads (“HpaII-beads” for simplicity) were washed again with Buffer A. Enrichments were performed either in 1.7 ml microcentrifuge tubes or in a 96-well plate. DNA samples suspended in 50 μl of Buffer B (10 mM Tris pH 8.0, 250 mM NaCl, 10 mM CaCl2, 0.01% Tween 20) were added to HpaII-beads and mixed for the indicated time. Magnetic beads were separated using either a tube magnetic stand (Life Technologies) or a plate magnet (Millipore, Billerica, MA). The beads were washed once with 200 μl Buffer A, and then resuspended in 50 μl of Buffer B for qPCR analysis.
For gel analysis and next-generation library preparation, the DNA was eluted from beads by incubation with 50 μl 5 M guanidinium thiocyanate at room temperature for 5 minutes. The eluent was transferred to a 3,500 MWCO dialysis tube (Thermo Scientific, Waltham, MA) and dialyzed against distilled water for 1 hour at room temperature.
For gel analysis and next-generation library preparation, the DNA was eluted from beads by incubation with 50 μl 5 M guanidinium thiocyanate at room temperature for 5 minutes. The eluent was transferred to a 3,500 MWCO dialysis tube (Thermo Scientific, Waltham, MA) and dialyzed against distilled water for 1 hour at room temperature.
8
Biotin-labeled DNA Pulldown Assay
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Biotin-labeled double strand DNA fragment (dsDNA, 500 bp) was generated using biotin-11-ddUTP (Thermo Scientific, #R0081) incorporation, and PCR amplification using Taq polymerase and a pMBP vector (as template). Biotin-labeled DNA (produced as above) or biotin dA-dT (70 mer) was conjugated on streptavidin magnetic beads (New England Biolabs, #S1420S) and incubated in Xenopus egg extracts and HeLa cell lysates. The beads were re-isolated using a magnet, washed five times, and then resolved by SDS-PAGE.
9
CRISPR-Cas9 RNA Synthesis Protocol
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S. pyogenes Cas9 (# M0386M), EnGen sgRNA Synthesis Kit, S. pyogenes (# E3322S), HiScribe T7 High Yield RNA Synthesis Kit (E2040S), 2× RNA Loading Dye (# B0363S), Nucleoside Digestion Mix (M0649S), Q5 Hot Start High-Fidelity 2× Master Mix (# M0494L), Monarch PCR & DNA Cleanup Kit (# T1030L), Proteinase K (# P8107S), Shrimp Alkaline Phosphatase (# M0371S), T4 Polynucleotide Kinase (# M0201S), Streptavidin Magnetic Beads (# S1420S), and NEBuffer 3.1 (# B7203S) with a 1× composition of 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μg/ml BSA, pH 7.9 at 25°C were all from New England Biolabs. Both S. pyogenes and S. aureus Cas9 were purified at New England Biolabs using standard liquid chromatography protein purification techniques. Protein stock concentration for both Spy- and SauCas9 was measured by absorbance of 280 nm light on a NanoDrop instrument (A280) as well as Bio-Rad Bradford assays per manufacturer protocol. All DNA oligomers were ordered from Integrated DNA Technologies. The Zymo RNA Clean & Concentrator-5 kit (#R1016) was purchased from Zymo Research. The SequaGel–UreaGel (# EC-833) system was from National Diagnostics. Yeast tRNA (# AM7119), 6% (# EC62652), and 15% (# EC6885) Tris-Borate EDTA gels were from Invitrogen/Thermo Fisher Scientific. γ-32P-ATP was from PerkinElmer (# BLU002A100UC).
10
Assembly of Pol II Elongation Complex
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Pol II elongation complex (EC10) was assembled essentially as previously described (29 (link)). First, radiolabeled 10-mer RNA was annealed to the TS DNA, followed by incubation with Pol II for 10 min at room temperature (23°C) and then 2 min at 37°C. To this, biotin-labeled NTS DNA was added and incubated for 5 min at 37°C, followed by 20 min at room temperature (23°C). The assembled elongation complex was incubated with streptavidin magnetic beads (NEB) for 30 min at room temperature (23°C) and subsequently washed with elongation buffer (EB) (20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 40 mM KCl, 5 mM DTT). The immobilized elongation complex was ligated to the downstream slip-out DNA and washed two times with EB buffer. Next, Rpb4/7 was added to a final concentration of 5 μM and incubated for 15 min at 23°C to generate 12-subunit elongation complex. Then the 12-subunit elongation complex was washed three times with EB buffer to remove excess Rpb4/7.
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