°C, and 1/9 sample volume of 10% NP-40 and 300 mM DHPC were added to the supernatant at final concentration of 1% (vol/vol). After incubation on ice for 5 min, the lysate was centrifuged for 10 min at 20,000×g to pellet insolubilized material. Then 200 μL of freshly resuspended affinity matrix was added to the supernatant and incubated at 4 °C for 16–18 h with gentle end-over-end mixing in a tube rotator. After incubation, the beads were collected with a magnet and resuspended in 1000 μL of high-salt buffer (20 mM HEPES KOH (pH 7.3), 350 mM KCl, 10 mM MgCl2, 1% NP-40, 0.5 mM DTT, and 100 μg/mL cycloheximide) and collected with magnets as above. After four times of washing with high-salt buffer, RNA was extracted using Absolutely RNA Nanoprep Kit (Agilent Technologies) following the manufacturer’s instructions. RNA quantification was measured using Qubit RNA HS Assay Kit (Life Technologies) and the integrity was determined by Bioanalyzer 2100 using an RNA Pico chip (Agilent Technologies). The cDNA library was prepared with Clontech SMARTer and then sequenced by HiSeq3000. Single-end reads of 50 base pairs were generated for an average of 29.2 million reads per sample (24 samples).
