T3 t7 rna polymerase

To identify linc1281-interacting proteins in mESCs, biotin-RNA pull down and subsequent liquid chromatography-mass spectrometry were performed. Briefly, biotin-labeled RNAs were synthesized by using RNA Labeling Mix (Roche, 11685597910) and T3/T7 RNA polymerase (Roche, 10881767001/11031171001). Linc1281 or antisense RNAs were heated to 90°C for 2 min, held on ice for 2 min and then incubated in room temperature for 20 min in RNA structure buffer (10 mM Tris–HCl pH 7.0, 0.1 M KCl and 10 mM MgCl₂) to form proper secondary structure. mESCs (1 × 10⁷ cells) were lysed with RIP lysis buffer and Immunoprecipitation was performed by using streptavidin beads coated with 3 μg of linc1281 or antisense RNAs. The RNA-binding proteins were sequenced and identified by LC–MS as described previously (41 (link)). The mass spectra were searched using the Mascot Daemon software (Version 2.3.0, Matrix Science, London, UK) based on the Mascot algorithm. The database used to search was the Mouse UniProtKB database.