To identify the binding partners of LncHrt, we used a tagged-RNA pulldown assay, as described [37 (link), 64 (link)]. Sense and antisense strands of streptavidin-binding S1m DNA were synthesized (Tsingke), annealed, and cloned into pCDH-MSCV at a cloning site, which was then used to insert sequences encoding LncHrt. The constructs expressing S1m, S1m-LncHrt as well as those expressing untagged LncHrt and EGFP were packaged into lentivirus using PEI (1:4) and then infected the cardiac fibroblasts with 20 MOI for 48 h. Cells were then harvested in SA-RNP lysis buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 2 mM DTT, 50 U/mL RNase OUT (Life Technologies) 50 U/mL Superase IN (Ambion) and 1 × complete protease inhibitor tablet (Roche). Streptavidin–Sepharose beads were blocked with 500 ng/µL yeast tRNA and 1 mg/mL BSA in SA-RNP lysis buffer before being added into cell lysates and being incubated at 37 °C for 2 h on a rotator. The beads were then pelleted and washed five times with SA-RNP washing buffer (20 mM Tris–HCl (pH 7.5), 300 mM NaCl, 5 mM MgCl2, 2 mM DTT, 50 U/mL RNase OUT (Life Technologies, NY, USA), 50 U/mL Superase IN (Ambion) and 1 × complete protease inhibitor tablet (Roche). After the last wash, RNA-bound proteins were then boiled in 50 µL 3 × LDS sample buffer (Life Technologies) and used for mass spectrometry.
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