ChIRP-MS Protocol for Identifying lncRNA Gm2044 Interactome

For ChIRP-MS, the ovarian cell suspension was reversibly cross-linked in situ with formaldehyde (Sangon Biotech, Shanghai, China), followed by cleaving with lysis buffer (50 mM Tris-HCl pH 7.0 (Sigma-Aldrich, St. Louis, MO, USA), 1% SDS (Sangon Biotech), 10 mM EDTA (Sigma-Aldrich), 1 mM PMSF (Sangon Biotech)) and hybridization with biotin-labeled RNA probes (control probe and Gm2044 probe). After the non-specific binding proteins were washed out under strong denaturation conditions, the obtained RNA-binding proteins were identified and quantitatively analyzed by liquid chromatography-tandem MS. Then, the interactive protein profile of lncRNA Gm2044 was constructed and analyzed with gene ontology (GO, http://geneontology.org/), the Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg/) and STRING (https://cn.string-db.org/). The probes of lncRNA Gm2044 were listed in Table S3, and the control probe was described in a previous study [17 (link)]. For RNA IP, the corresponding RNA-protein complexes were precipitated using antibodies against EEF2 protein (ABclonal Technology, Wuhan, China), and the purified protein and RNA were then analyzed according to the protocol in previous studies [18 (link), 19 (link)].