Pull-down assay was carried out as described.21 (link) Biotinylated miRNA (Bio-NC, Bio-miR-1271 WT or Bio-miR-1271 MUT) was transfected into Huh7-1.3 and HepAD38 cells. Cells were collected after 48 h and then lysed with lysis buffer. Cells lysate was incubated with MyOne C1 Dynabeads (Invitrogen) and then washed by lysis buffer. After elution, the RNA level was assessed by qRT-PCR.
Dynabeads myone c1
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dynabeads MyOne C1 are uniform, superparamagnetic beads with a carboxylated surface. They are designed for applications that require the capture and isolation of target molecules from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Laemmli buffer, by Merck Group (5 mentions)
Superase in, by Thermo Fisher Scientific (3 mentions)
Yeast trna, by Thermo Fisher Scientific (3 mentions)
Sheared salmon sperm dna, by Thermo Fisher Scientific (3 mentions)
Igepal ca 630, by Merck Group (3 mentions)
Nupage novex bis tris precast gel, by Thermo Fisher Scientific (3 mentions)
Ribomax large scale rna production system, by Promega (2 mentions)
Microspin g 50 column, by GE Healthcare (2 mentions)
T4 ligase, by Thermo Fisher Scientific (2 mentions)
Desthiobiotin datp, by Jena Biosciences (2 mentions)
T4 kinase, by Thermo Fisher Scientific (2 mentions)
Dna polymerase, by Thermo Fisher Scientific (2 mentions)
Pcsk9 pc9 h5223, by ACROBiosystems (2 mentions)
Il 6 il6 h4218, by ACROBiosystems (2 mentions)
Aminolink plus micro immobilization kit, by Thermo Fisher Scientific (2 mentions)
T3 dna ligase, by New England Biolabs (2 mentions)
T4 rna ligase buffer, by New England Biolabs (2 mentions)
Hbs p, by GE Healthcare (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
21 protocols using dynabeads myone c1
1
Identifying miRNA-mRNA Interactions Using Pull-down Assay
2
Nascent RNA Labeling and Capture
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The ability to capture nascently transcribed RNA was modified from previously described work (Payne et al., 2014 (link)). In brief, cells were treated with 200 µM 4-thiouridine (4sU) for one hour before total RNA was harvested in 1 mL of TRIzol (Life Technologies). Nascently transcribed RNA over this time period incorporated 4sU, which was subsequently biotinylated and pulled down using streptavidin MyOne C1 Dynabeads (Invitrogen). RNA was reverse transcribed and qPCR was performed as described above.
3
Biotinylated miRNA Pull-Down Assay
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Biotinylated-miRNA pull-down assay was performed as described previously [31 (link)]. Briefly, H4-APPswe cells were transfected with 75 nM biotinylated-miR-1273g-3p or biotinylated cel-miR-39-3p as a negative control (Exiqon). After 24 h, cells were lysed with hypotonic buffer containing 10 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 0.5% NP40, 50 U/mL SUPERaseIn (Invitrogen) and 1x protease inhibitor cocktail (Roche). The supernatants were transferred to 25 μL of streptavidin-containing myOne C1 Dynabeads (Invitrogen) which were pre-blocked with 1 μg/μL BSA and MS2 RNA (Roche), and the same volume of 2 M NaCl hypotonic buffer as the lysate was added. The mixture was incubated for 30 min with gentle rotation and the beads were washed with 1 M NaCl hypotonic buffer. Total RNA was extracted using miRNeasy micro kit (Qiagen) according to the manufacturer’s instruction.
4
Capturing and Washing Biotinylated Oligos
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The ligation mixture was diluted by adding an equal volume of water to reduce the viscosity of the solution. Next, streptavidin-coated MyOne C1 Dynabeads (Thermo Fisher) were added to each sample in a 1.2:1 excess over CHO (for example, a 50 µL reaction had 50 pmol capture hairpin oligo; beads were supplied at 10 mg/ml and had a binding capacity of 500 pmol biotinylated oligo per mg, so use 12 µL beads). The bead-sample mixture was incubated at room temperature for 15 min. After binding, supernatants were removed, and the beads were washed once with high salt wash buffer (1 M NaCl, 20 mM Tris-HCl, pH 7.4) and once with low salt wash buffer (100 mM NaCl, 20 mM Tris-HCl, pH 7.4). After washing, multiple individually barcoded samples can be combined for downstream steps. At this stage, enzymatic or chemical treatments can be incorporated into the library preparation protocol such as AlkB demethylase reaction or CMC treatments (see below).
5
Isolation and Elution of RNA Targets
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50µl of MyONE C1 Dynabeads (Thermo Fisher; Waltham, MA, USA) were mixed with 250pmol of oligos (Sequences: Table S1) biotinylated at the 5' end corresponding to either the responsive (R; positions 5-54) or non-responsive (nR; positions 55-104) regions (Integrated DNA Technologies; Leuven, Belgium) for 12 min at 37¡C with gentle rotation. Beads were then washed in WB (5mM Tris-HCl pH 7.5, 0.5mM EDTA, 1M NaCl, 0.05% Tween) and resuspended in 50µl NT buffer. Lysates were extracted from MCF7 cells by resuspending in PLB as above, in the presence or absence of Queretin at 10µM, or after transfection with siHuR or siControl. 850µl of NT buffer was added to 100µl cell lysate in PLB, and 50µl set aside for input sample. 50µl of beadoligo complex was added and the mixture incubated for 2 h at room temperature with end-over-end rotation. Beads were washed three times in NT buffer, and resuspended in 20µl TE (1mM EDTA pH 7.5, 10mM Tris-HCl pH 7.5). This was heated to 70¡C at 0.5¡C/s, and then immediately allowed to return to room temperature. Supernatant containing the eluted RNA was resuspended in miRVana buffer.
6
Oligonucleotide Synthesis and Purification
7
Isolation and RNA Extraction from Dynabeads
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A volume of 25 μl of MyOne C1 Dynabeads (LifeTech) was added per sample. Beads had been previously washed with DEPC-treated 0.1 M NaOH, 0.05 M NaCl (twice bead volume), and then washed once in DEPC-treated 0.1 M NaCl and once in hypotonic lysis buffer. Beads were blocked with 1 μg/μl bovine serum albumin and 1 μg/μl yeast tRNA prepared in hypotonic lysis buffer and rotated for 3 h at 4 °C. Samples and beads were incubated for 30 min with rotation and then placed on a magnetic stand and the remove supernatant removed. The beads were washed three times with 200 μl of hypotonic lysis buffer. RNA was extracted from the beads using a standard Trizol extraction, and RNA was reverse transcribed into cDNA for qPCR analysis. Data analysis was compared to % of input controls.
8
RNA Affinity Purification of Proteins
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RNA probe was generated by RiboMAX™ Large Scale RNA Production Systems (Promega) using DNA template containing 5′-T7 promoter, sequence of interest and 3′-aptamer. A total of 25 µg RNA probe was incubated with 50 µl Dynabeads MyOne C1 (Invitrogen) in 300 µl binding buffer (100 mM NaCl, 10 mM MgCl2, 50 mM Hepes, pH 7.4, and 0.5% Igegal CA-630) for 30 min at 4 °C with rotation followed by three washes with washing buffer (250 mM NaCl, 10 mM MgCl2, 50 mM Hepes, pH 7.4, and 0.5% Igegal CA-630). For each reaction, 400 µg nuclear extract or 1 mg whole cell extract was diluted in 300 µl washing buffer and supplemented with 4 µl 10 mg/ml yeast tRNA (Invitrogen) and SUPERase In (Invitrogen). RNA immobilized beads were incubated with protein mixtures for 30 min at 4 °C or room temperature with rotation. After three washes, bound proteins were eluted in 2× Laemmli buffer (Sigma) at 95 °C and analyzed by western blot (WB). Sequences of probes are listed in Supplementary Table 2 .
9
RNA Probe Binding and Protein Extraction
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RNA probe was generated as discussed above but only with an addition of 3′-aptamer at reverse primer. For each reaction, 50 µl Dynabeads MyOne C1 (Invitrogen) was used to incubate with 25 µg RNA probe in 300 µl binding buffer (100 mM NaCl, 10 mM MgCl2, 50 mM Hepes, 0.5% Igegal CA-630, and pH 7.4) for 30 min at 4 °C with rotation for probe binding to the beads. Beads were then washed washing buffer (250 mM NaCl, 10 mM MgCl2, 50 mM Hepes, 0.5% Igegal CA-630, and pH 7.4,) for 10 min at 4 °C for three times. 1 mg whole cell extract was supplemented with 4ul 10 mg/ml yeast tRNA (Invitrogen) and SUPERase In (Invitrogen). The mixture was added to the beads and topped up to 300 µl with washing buffer, followed by incubation for 30 min at 4 °C with rotation. After washing three times, beads were subjected to 2× Laemmli buffer (Sigma) at 95 °C to elute proteins followed by western blot (WB). Sequences of probes are listed in Supplementary Data 5 .
10
Affinity Capture of Transcription Factors
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The 1.5Kb upstream region at Nos2 or Il6 (negative control) promoter was amplified with 5’‐biotinylated forward primer and 5’‐unmodified reverse primer (Table S1). Nuclear fraction of macrophages were lysed by 90 min incubation in two volumes of nuclear lysis buffer (420 mM NaCl, 20 mM HEPES pH 7.9, 20% v/v glycerol, 2 mM MgCl2, 0.2 mM EDTA, 0.1% NP40, protease, and RNase inhibitor and 0.5 mM DTT), and sonicated to improve lysis. 5‐biotinylated promoters were immobilized on Dynabeads MyOne C1 (Invitrogen, Waltham, Massachusetts, USA) by incubating for 30 min at room temperature in binding buffer (1 M NaCl, 10 mM Tris‐HCl pH 8, 1 mM EDTA pH 8, and 0.05% NP‐40). Beads containing immobilized probes were then incubated with nuclear extracts of macrophages in protein binding buffer (50 mM Tris‐HCl pH8, 150 mM NaCl, 1 mM DTT, 0.25% NP‐40 and complete protease and RNase inhibitors) for 30 min at 37°C and another 2 h at 4°C in the presence of poly‐dAdT (Sigma‐Aldrich, St. Louis, Missouri, USA). Complex‐containing beads were washed extensively (using incubation buffer without poly‐dAdT), and RNA fractions were extracted using the Trizol reagent.
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