Streptavidin dynabeads

Biotin-labelled lncRNA-HGBC was first transcribed in vitro from pBluescript II SK-lncRNA-HGBC using Biotin RNA Labeling Mix (Roche, Germany) by SP6(for anti-sense)/T7(for sense) RNA polymerase (Roche) according to the manufacturer’s instructions. The RNA products were treated with RNase-free DNase I (Roche) and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA). Four microgram biotinylated RNAs were denatured for 5 min at 65 °C in PA buffer (10 mM Tris HCl pH 7.5, 10 mM MgCl2, 100 mM NH4Cl) and slowly cooled down to room temperature. Then, the folded RNA was incubated with streptavidin Dynabeads (Invitrogen) for 1 h at 4 °C in the presence of 2 U/ml RNasin (Promega). After washing 4 × 5 min with wash buffer (10 mM HEPES pH 7.0, 400 mM NaCl, 1 mM DTT, 1% Triton X-100, protease inhibitor cocktail (Roche), 2 mM RVC), the protein lysate from 1 × 10⁷ NOZ cells was pre-cleared by streptavidin Dynabeads (Invitrogen) and incubated with the folded RNA-beads complex for 3.5 h at 4 °C in the presence of 20 μg/ml yeast tRNA. After extensive washing, beads were boiled 40 μl of 1× SDS loading buffer for 10 min at 100 °C. The lncRNA-interacting proteins were further separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and the gel was silver stained. Then, lncRNA-HGBC specific bands were subjected to mass spectrometry and retrieved in human proteomic library.

Plasmid of pcDNA3.1-PAS1 was linearized as DNA templates. Biotin-labeled RNA was produced with the MEGAscript T7 Kit (Ambion) with biotin-16-UTP (Ambion). The biotin-labeled RNA was further purified using the MEGA clear Kit (Ambion). 10 μg biotin-labeled RNA in RNA structure buffer (10 mM Tris pH7, 0.1 M KCl, 10 mM MgCl₂) was heated to 95 °C for 2 min, on ice for 3 min, and then at RT for 30 min. Then RNA was incubated with pre-cleared cell lysates (containing 1 mg proteins) in 500 μl binding buffer (150 mM KCl, 25 mM Tris pH 7.4, 0.5% DTT, 0.5% NP40, 1 mM EDTA, 2 U/ml RNasin) and then incubated at RT for 1 h. Next, 25 μl of washed streptavidin Dynabeads (Invitrogen) was added to each binding reaction and incubated at RT for 1 h. The streptavidin Dynabeads were washed with binding buffer for 5 times and boiled in SDS buffer. The retrieved proteins were analyzed by Western blot.

Expression of MBP fusion proteins was induced in BL21 cells with 1 mM IPTG at an optical density at 600 nm of 0.4–0.6 for 2 hr at 37°C. Cells were pelleted and lysed in 1x BugBuster Protein Extraction Reagent (Millipore Sigma 70584) for 20 min at 23°C. Cell lysates were centrifuged at 16,000xg at 4°C for 20 min to remove cellular debris.
MBP fusion proteins were batch purified from cleared lysates with amylose resin (New England Biolabs E8021S) in 1x amylose binding buffer (20 mM Tris-HCl pH7.4, 200 mM NaCl, 1 mM EDTA) overnight at 4°C. Resin was washed 10 times with 1x amylose binding buffer with 1% Triton X-100 and 0.5% deoxycholate. Purified MBP fusion proteins were batch eluted in 1x amylose elution buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM EDTA, 10 mM maltose).
Purified MBP fusion proteins were captured with biotin-peptide coated Streptavidin Dynabeads (Thermo Fisher Scientific 11047) overnight at 4°C in 1x PBS. The next day, Streptavidin Dynabeads were washed 10 times in 1x RIPA buffer, and captured proteins, along with input controls, were resolved by SDS-PAGE and visualized by Coomassie Blue staining.