Nanodrop onec

The PCR products were transcribed for 6 h at 37 °C using MegaScript kit (Ambion, Kaufungen, Germany), digested for 5 min at 37 °C with TURBO DNAse (Ambion) and purified using MegaClear kit (Ambion). The purified transcription products were precipitated for 16 to 18 h at −20 °C in 100% EtOH, 10% (v/v) 3.0 M sodium acetate (pH 5.3), and resuspended in RNAse-free water [20 (link)]. RNA concentration and purity were assessed using NanoDrop OneC and the profiles were observed after denaturing gel electrophoresis using a 10% Mini-PROTEAN^® TBE-Urea Precast gel (Bio-Rad, Hercules, CA, USA) or using the Experion automated electrophoresis system with the RNA StdSens analysis kit (Bio-Rad) after a denaturation at 90 °C for 2 min followed by 5 min in ice.

Cells were lysed in EBC buffer (50 mM Tris, pH 7.5, 120 mM NaCl, 0.5% NP-40) or Triton X-100 buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The protein concentrations of whole cell lysates were measured by NanoDrop OneC using the Bio-Rad protein assay reagent according to manufacture instructions^{13 (link)}. Equal amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitations analysis, 1000 μg lysates were incubated with the indicated antibody (1–2 μg) for 3–4 h at 4 °C followed by 1 h incubation with 10 μl Protein A magnetic beads (New England Biolabs). Or 1000 μg lysates containing tagged molecules were incubated with agarose beads coupled antibodies for the specific tag for 3–4 h at 4 °C. The recovered immuno-complexes were washed five times with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.5% NP-40) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies. Uncropped images are provided in Supplementary Fig. 14.

Cells were lysed in EBC buffer (50 × 10⁻³m Tris pH 7.5, 120 × 10⁻³m NaCl, 0.5% NP‐40) or Triton X‐100 buffer (50 × 10⁻³m Tris pH 7.5, 150 × 10⁻³m NaCl, 1% Triton X‐100) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail sets I and II, Calbiochem). The protein concentrations of whole cell lysates were measured by NanoDrop OneC using the Bio‐Rad protein assay reagent as described previously.^[55] Equal amounts of whole cell lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotted with indicated antibodies. For immunoprecipitation analysis, unless specified, 1000 µg lysates were incubated with the indicated antibody (1–2 µg) for 3–4 h at 4 °C followed by 1 h incubation with 10 µL Protein A magnetic beads (New England Biolabs). Or, 1000 µg lysates containing tagged molecules were incubated with agarose‐bead‐coupled antibodies for the specific tag for 3–4 h at 4 °C. For endogenous IPs, incubation of cell lysates with antibodies was extended to overnight. The recovered immunocomplexes were washed 5 times with NETN buffer (20 × 10⁻³m Tris, pH 8.0, 100 × 10⁻³m NaCl, 1 × 10⁻³m ethylenediaminetetraacetic acid (EDTA), and 0.5% NP‐40) before being resolved by SDS‐PAGE and immunoblotted with indicated antibodies.