Incubation:Article Title: MicroRNAs Cause Accelerated Decay of Short-Tailed Target mRNAs
Article Snippet: .. Following the last wash, beads were resuspended in an initial bead volume of 1X high salt wash buffer (HSWB, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 M NaCl, 0.01% Tween-20) supplemented with 0.5 μg/mL yeast RNA (ThermoFisher) and incubated at room temperature for 30 min with end-over-end rotation, again using a volume equal to that of the initial bead suspension. ..
Article Title: The Dynamics of Cytoplasmic mRNA Metabolism
Article Snippet: .. Following the last wash, beads were resuspended in an initial bead volume of 1X high salt wash buffer (HSWB, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 M NaCl, 0.01% Tween-20) supplemented with 0.5 mg/mL yeast RNA (ThermoFisher) and incubated at room temperature for 30 min with end-over-end rotation, again using a volume equal to that of the initial bead suspension. ..
Article Title: MicroRNAs Cause Accelerated Decay of Short-Tailed Target mRNAs
Article Snippet: .. Following the last wash, beads were resuspended in an initial bead volume of 1X high salt wash buffer (HSWB, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 M NaCl, 0.01% Tween-20) supplemented with 0.5 mg/mL yeast RNA (ThermoFisher) and incubated at room temperature for 30 min with end-over-end rotation, again using a volume equal to that of the initial bead suspension. ..
Article Title: Lysosomal RNA profiling reveals targeting of specific types of RNAs for degradation
Article Snippet: .. Following the last wash, beads were resuspended in an initial bead volume of 1X high salt wash buffer (HSWB, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 M NaCl, 0.01% Tween-20) supplemented with 0.5 mg/mL yeast RNA (Thermo Fisher) and incubated at 23°C on a thermal mixer, shaking in a cycle of 15 s 1400 RPM on and 1 min 45 s off, for 30 min, again using a volume equal to that of the initial bead suspension. .. Beads were then washed three times with 1X HSWB per reaction and split for each reaction during the last wash. After the wash was removed, sample RNA previously resuspended in 100 μL 1X HSWB was added to blocked beads and incubated at 23°C on a thermal mixer, shaking in a cycle of 15 s 1400 RPM on and 1 min 45 s off, for 30 min. Beads were washed twice with 400 μL water at 50°C, incubating at 50°C for 2 min for each wash, and then twice with 400 μL 10X HSWB, incubating at 50°C for 2 min for each wash. RNA was eluted from beads by incubating with 180 μL 0.5 M tris(2-carboxyethyl)phosphine (TCEP, Sigma-Aldrich) at 50°C shaking on a thermal mixer in a cycle of 15 s 1400 RPM on and 1 min 45 s off, for 30 min.
Suspension:Article Title: MicroRNAs Cause Accelerated Decay of Short-Tailed Target mRNAs
Article Snippet: .. Following the last wash, beads were resuspended in an initial bead volume of 1X high salt wash buffer (HSWB, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 M NaCl, 0.01% Tween-20) supplemented with 0.5 μg/mL yeast RNA (ThermoFisher) and incubated at room temperature for 30 min with end-over-end rotation, again using a volume equal to that of the initial bead suspension. ..
Article Title: The Dynamics of Cytoplasmic mRNA Metabolism
Article Snippet: .. Following the last wash, beads were resuspended in an initial bead volume of 1X high salt wash buffer (HSWB, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 M NaCl, 0.01% Tween-20) supplemented with 0.5 mg/mL yeast RNA (ThermoFisher) and incubated at room temperature for 30 min with end-over-end rotation, again using a volume equal to that of the initial bead suspension. ..
Article Title: MicroRNAs Cause Accelerated Decay of Short-Tailed Target mRNAs
Article Snippet: .. Following the last wash, beads were resuspended in an initial bead volume of 1X high salt wash buffer (HSWB, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 M NaCl, 0.01% Tween-20) supplemented with 0.5 mg/mL yeast RNA (ThermoFisher) and incubated at room temperature for 30 min with end-over-end rotation, again using a volume equal to that of the initial bead suspension. ..
Article Title: Lysosomal RNA profiling reveals targeting of specific types of RNAs for degradation
Article Snippet: .. Following the last wash, beads were resuspended in an initial bead volume of 1X high salt wash buffer (HSWB, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 M NaCl, 0.01% Tween-20) supplemented with 0.5 mg/mL yeast RNA (Thermo Fisher) and incubated at 23°C on a thermal mixer, shaking in a cycle of 15 s 1400 RPM on and 1 min 45 s off, for 30 min, again using a volume equal to that of the initial bead suspension. .. Beads were then washed three times with 1X HSWB per reaction and split for each reaction during the last wash. After the wash was removed, sample RNA previously resuspended in 100 μL 1X HSWB was added to blocked beads and incubated at 23°C on a thermal mixer, shaking in a cycle of 15 s 1400 RPM on and 1 min 45 s off, for 30 min. Beads were washed twice with 400 μL water at 50°C, incubating at 50°C for 2 min for each wash, and then twice with 400 μL 10X HSWB, incubating at 50°C for 2 min for each wash. RNA was eluted from beads by incubating with 180 μL 0.5 M tris(2-carboxyethyl)phosphine (TCEP, Sigma-Aldrich) at 50°C shaking on a thermal mixer in a cycle of 15 s 1400 RPM on and 1 min 45 s off, for 30 min.
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