Immobilized trypsin

To identify H19-binding proteins, H19 pulldown was performed as previously described [21 (link)]. Briefly, biotin-labeled H19 RNAs were in vitro transcribed with Biotin RNA Labeling Mix (Roche) and MEGAscript® Transcription Kit (Ambion) then further purified with RNA Clean & Concentrator-5 (Zymo Research). Human skeletal muscle tissues (Additional file 2: Table S2) and mouse skeletal muscle tissue lysates were prepared using the RIPA buffer with anti-RNase, protease/phosphatase inhibitor cocktails supplemented in the lysis buffer. The eluted RNA-protein complexes were denatured, reduced, alkylated, and digested with immobilized trypsin (Promega) for LC-MS analysis at the MD Anderson Cancer Center Proteomics Facility. In vitro RNA-protein binding assay and in vitro RNA pull-down coupled with dot-blot assays were performed as previously described [22 (link)]. Briefly, the RNA-capture beads were incubated with recombinant DMD (aa. 3046–3685) protein in binding buffer [50 mM Tris-HCl pH 7.9, 10% Glycerol, 100 mM KCl, 5 mM MgCl₂, 10 mM β-ME, 0.1% NP-40] for 1 h at 30 °C. Post proteinase K digestion, the RNA fragments were hybridized to the dot-blot with probes reverse complimentary to the human H19 sequence. Dot-blot probe sequences are listed in the Additional file 1: Table S1.

LncRNA 91H RNAs in vitro were transcribed using the Biotin RNA Labeling Mix (Roche) and SP6 RNA polymerase (Ambion) and purified by RNA Clean & Concentrator™-5 (Zymo Research). Then cell lysates were freshly prepared using Protea Prep Zwitterionic Cell Lysis Kit, Mass Spec Grade (Protea^®) with Anti-RNase, Protease/Phosphatase Inhibitor Cocktail, Panobinostat and Methylstat supplemented in the lysisbuffer. The BcMag™ Monomer avidin Magnetic Beads (Bioclone) were firstly prepared according to manufacturer’s instructions and then immediately subjected to RNA (20 µg) capture in RNA capture buffer for 25 min at room temperature with agitation. The RNA-captured beads were washed with NT2 buffer and incubated with 30 mg cell lysates diluted in NT2 buffer supplemented with 50 U/ml RNase OUT™, 50 U/ml Superase1·IN™, 2 mM dithiothreitol, 30 mM EDTA and Heparin 0.02 mg/ml at 4 °C for 2 h with rotation. Washing with NT2 buffer, NT2-high salt buffer (500 mM NaCl), NT2-high salt buffer (1 M NaCl), NT2-KSCN buffer (750 mM KSCN) for twice and PBS (once) for 5 min at 4 °C and eluted by 2 mM D-biotin in PBS. The eluted protein complexes were denatured, reduced, alkylated and digested with immobilized trypsin (Promega) for MS analysis at MD Anderson Cancer Center Proteomics Facility.

Hct-116 Cells treated with SARS-COV-2 N plasmid and negative control was harvested and extracted total proteins. Then, the protein concentration was quantified using a BCA protein assay kit (Beyotime, Shanghai, China). Western-blot was further used to separate the proteins. Then the N protein complexes were denatured, reduced, alkylated and digested with immobilized trypsin (Promega) for mass spectrometry analysis.