Megascript t7 transcript kit

The RNA pulldown assays were performed to identify proteins interacting with LINC00240 as reported previously [15 (link), 23 (link)]. To prepare the DNA template for in vitro RNA synthesis, LINC00240 was subcloned into pcDNA3.1 with inserted T7 promoter before and after the cloning site. In briefs, LINC00240 RNAs were transcribed with T7 RNA polymerase (MEGAscript T7 Transcript Kit, Thermo fisher, AM1330) and the linearized LINC00240 construct. After purified with the RNeasy minikit (Qiagen, #74,104, Germany), the sense and antisense LINC00240 RNAs were biotinylated with Pierce™ RNA 3' End Desthiobiotinylation Kit (Thermo fisher, 20,163). These RNAs were then incubated with MGC80-3 protein extracts at 4 °C for 1 h. Proteins bound on the streptavidin magnetic beads were recovered with Elution Buffer following the instruction of Pierce™ Magnetic RNA–Protein Pull-Down Kit (Thermo, 20,164, USA). The retrieved proteins were then analyzed by liquid chromatography-tandem mass spectrometry (LS-MS/MS) (Hoogen Biotech Co., Shanghai, China) and Western Blot. Mass spectra were analyzed using MaxQuant software (version 1.5.3.30) with the UniProtKB human database (Uniport Homo sapiens 188441_20200326).

RNA pulldown was performed following the standard protocol as reported previously.^{23 (link),48 (link)–50 (link)} To prepare the plasmid construct template for in vitro RNA synthesis, LCETRL3 or LCETRL4 was subcloned into pcDNA3.1 with inserted T7 promoter before and after the cloning site. After these constructs were linearized, lncRNAs were transcribed with T7 RNA polymerase (MEGAscript T7 Transcript Kit, Thermo fisher, AM1330) and purified with the RNeasy minikit (Qiagen, #74104, Germany). Pierce™ RNA 3’ End Desthiobiotinylation Kit (Thermo fisher, 20163) was used to biotinylate sense and antisense LCETRL3 or LCETRL4 RNAs. These RNAs were then incubated with PC9 or H1299 protein extracts at 4 °C for 1 h. Proteins bound on the streptavidin magnetic beads were eluted with the Elution Buffer of Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo, 20164). The recovered proteins were then analyzed by liquid chromatography-tandem mass spectrometry (LS-MS/MS) (Hoogen Biotech Co., Shanghai, China) and Western Blot. MaxQuant software (version 1.5.3.30) with the UniProtKB human database (uniport Homo sapiens 188441_20200326) was utilized to analyze mass spectra.