Dyna protein g beads

PAR-CLIP was performed as previously described (Garzia et al., 2017 ; Hafner et al., 2010 (link)) with minor adjustments. In brief, 3–5 × 10⁹ THP-1 cells were doxycycline induced and labeled with 100 μM 4SU 16 hours before harvesting and UV_365nm irradiation. Stimulated THP-1 cells were additionally treated with EC₅₀ cyclic GMP-AMP 16 hours before harvest. After crosslinking, THP-1 cells were lysed using NP-40 lysis buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 2 mM EDTA, 1 mM NaF, 2% (v/v) NP-40, 0.5 mM DTT, Roche EDTA-free protease inhibitor) and incubated with Dyna-protein G beads (Invitrogen) coupled with anti-FLAG M2 antibody (Sigma) for 2 hours at 4°C. Beads were washed with high-salt buffer and then underwent CIP and T4 PNK mediated 5′ end RNA radiolabeling with [γ−³²P]-ATP. Flag-tagged ELAVL1 crosslinked to RNA was then was resolved on a 4%–20% Bis-Tris, NuPage gradient gel (Invitrogen). The band corresponding to ELAVL1 protein was cut out. The protein: RNA complex was then electroeluted out of the gel and treated with proteinase K (Roche). RNA was then size-selected and underwent both 3′ (MultiplexDX Inc.) and 5′ adaptor (Illumina compatible) ligation and was reverse transcribed into cDNA. cDNA library was sequenced on the NextSeq Illumina platform at Hudson Alpha.