Biotin rna labelling mix

To prepare the biotin-labeled RNA, 2 ug plasmid pcDNA3.1 α1AT were first linearized with NheI, purified and then transcripted by T7 RNA polymerase (NEB, Ipswich, MA, USA) with Biotin-RNA labelling Mix (Roche, Basel, Switzerland). The Biotin labelled RNA was then digested with RNase-free DNase and purified with RNA Cleanup Kit (Qiagen, Hilden, Germany). For biothylated RNA pull down, C3A cells were first lysed by extraction buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5% glycerol, 0.1% Triton X-100, 1 mm DTT, 1 mm PMSF and protease Inhibitor) the nuclear and debris were pelleted by centrifugation at 13,000 rpm for 20 min. In each assay 2 μg of biotinylated transcript were introduced into 500 ug of C3A cell extract incubated and shaking in 4 °C for 1 h; 20 μL of washed Streptavidin agarose beads (Invitrogen, Carlsbad, CA, USA) were added to each binding reaction and further incubated at RT for 1 h. Beads were then washed briefly three times with lysis buffer and collected by centrifugation, and then were loaded on SDS-PAGE gels, and the corresponding band was sent for mass spectrometry analysis.

The biotin-labelled lncRNA (both wild type and mutant type) and the antisense RNA were in vitro transcribed with a Biotin RNA Labelling Mix (Roche, CA, USA) and the T7 RNA polymerase (Roche), treated with RNase-free DNase I (Roche) and purified with an RNeasy Mini Kit (Qiagen). CD4⁺T cell extracts were incubated with biotinylated RNAs and 60 μl of streptavidin agarose beads (Invitrogen Life Technologies). The associated proteins were resolved by SDS–polyacrylamide gel electrophoresis, and specific bands were excised. Proteins were eluted, digested and subjected to the OrbitrapVelos Pro LC/MS system (Thermo Scientific, CA, USA). Data were analysed by Proteome Discoverer and the resulting peak lists were used for searching the NCBI protein database with the Mascot search engine.

RNA was biotinylated using Biotin-RNA Labelling Mix (Roche Diagnostics International Ltd., Rotkreuz, Switzerland) and transcribed in vitro using T7 RNA polymerase (Roche, Basel, KB, Switzerland). After lysis with a cell lysis buffer, the cells were sonicated and centrifuged. Following this, biotinylated RNA molecules were incubated with a cell supernatant. Subsequently, magnetic beads were used for targeting RNAs, and the co-precipitated protein samples were detected by western blotting.

RNAs were labelled with SP6/T7 RNA polymerase (Roche) and the Biotin RNA labelling mix (Roche Diagnostics) in vitro and treated with RNase‐free DNase I (Roche). Then, RNeasy Mini Kit (Qiagen) was utilized for the purification of RNAs labelled with biotin (Bio‐miR‐488‐Mut, Bio‐miR‐488‐WT and Bio‐NC). Thereafter, the mixed solution (50 pmol of biotinylated RNA and 1 mg NSCLC cell) was added with washed beads (6 mL) containing streptavidin agarose (Life Technologies), and 1 hour of incubation was carried out at room temperature. After the rinsing of beads, the amplification of eluted RNAs was conducted, followed by measurement of them via qRT‐PCR.

RNA pulldown was performed as previous described [38 (link), 39 (link)]. Biotinylated PURPL transcript and EGFP RNAs were transcribed using a MAXIscript T7/T3 in vitro transcription kit (Ambion) and Biotin RNA labelling Mix (Roche).