Glycine

To prepare PKR antibody-conjugated beads, protein A beads (Thermo Fisher Scientific) was incubated with PKR antibody (Cell Signaling Technology) in the fCLIP lysis buffer (20 mM Tris-HCl, pH 7.5, 15 mM NaCl, 10 mM EDTA, 0.5% NP-40, 0.1% Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate) for 3 h at 4℃ after adjusting NaCl concentration to 150 mM. Harvested cells were fixed with 0.1% (w/v) filtered paraformaldehyde (Sigma Aldrich) for 10 min and immediately quenched by adjusting glycine (Bio-basic) concentration to 250 mM. The crosslinked cells were lysed using the fCLIP lysis buffer for 10 min on ice and then sonicated. The NaCl concentration of the lysate was adjusted to 150 mM, and cell debris was separated by centrifugation. The lysate was added to the PKR antibody-conjugated beads and incubated for 3 h at 4℃. The bead was washed 4 times with the fCLIP wash buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.1% NP-40, 0.1% SDS, 0.1% Triton X-100, and 0.1% sodium deoxycholate) and PKR-dsRNA complex was eluted from the beads by incubating in the elution buffer (200 mM Tris-HCl pH 7.4, 100 mM NaCl, 20 mM EDTA, 2% SDS, and 7 M Urea) for 3 h at 25℃. The eluate was treated with 20 mg/ml proteinase K (Sigma Aldrich) for overnight at 65℃. RNA was purified using acidphenol:Chloroform pH 4.5 (Thermo Fisher Scientific).