2.5. RNA immunoprecipitation (RIP) and quantitative RT-PCR

Fibroblasts were either treated with 0.5 mM sodium arsenite for 45 min or left untreated. Cells were washed with PBS and RNAs were crosslinked to proteins using UV crosslinker at 200 mJ/cm². Whole cell lysates were prepared by resuspending the cells in lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.5% NP-40, 0.5% sodium deoxycholate, 5 mM EDTA, 2 mM DTT, 0.5 U/μl RNase inhibitor and 1X Complete protease inhibitor), followed by centrifugation at 12,000 rpm for 10 min and collecting the supernatant. Five hundred μg cell lysate was used to pull-down DDX1-bound RNAs with anti-DDX1 antibody (batch 2910) [16,35] for 1.5 h at 4 °C followed by incubation with Protein A Sepharose beads (GE Healthcare) for another 1.5 h at 4 °C with end-to-end rotation. Rabbit IgG was used as the negative control. Unbound fractions were removed, and beads were washed with wash buffer (50 mM Tris-Cl pH7.5, 1 M NaCl, 1% NP-40, and 1% sodium deoxycholate). Any contaminating DNA was removed by incubating the beads with 12 units of Turbo DNase (Thermo Fisher Scientific) at 37 °C for 30 min. Bound RNAs were separated from beads by digesting the proteins with Proteinase K (Roche) at 37 °C for 30 min. RNAs were extracted with phenol:chloroform (1:1), ethanol precipitated and dissolved in 20 μl RNase-free water.

To prepare cDNA, 5 μl RNA eluted from the RNA-immunoprecipitations was reverse transcribed using Superscript IV reverse transcriptase (Thermo Fisher Scientific) and random hexamer primers. Quantitative RT-PCR was performed with SYBR Green qPCR mix (Applied Biological Materials Inc.) and the primers listed in Table S1. For normalization purposes, RNA was extracted from cells grown under the same conditions as those used for RNA immunoprecipitations. Total RNA was reverse transcribed and used to determine cellular levels of each RNA targets under each condition. RNA enrichment in RNA immunoprecipitations was normalized against individual RNA levels under the same conditions. GAPDH served as the internal control.