4sU-RNA-seq was performed as described in Rabani et al.60 (link). In brief, cells were incubated with the medium supplemented with 500 μM 4sU for 15 min, after which medium was rapidly removed and cells were lysed with 5 mL of Trizol reagent (Life Technologies). Total RNA was treated with Ambion DNA-free DNase Treatment kit (Life Technologies) and resuspended in water. For each μg of total RNA, 2 μL of Biotin-HPDP (Pierce, 50 mg EZ-Link Biotin-HPDP) dissolved in DMF at a concentration of 1 mg/mL, and 1 μL of 10X Biotinylation buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA), was added. After incubation for 15 min at 25 °C, the RNA was purified using two rounds of chloroform purification in Phase Lock Gel Heavy Tubes (Eppendorf), precipitation with an equal volume of isopropanol and 1/10 volume of 5 M NaCl, and finally resuspended in water.
Biotinylated 4sU-RNA was recovered using the μMacs Streptavidin Kit (Miltenyi). Per microgram of recovered biotinylated 4sU-RNA, 0.5 μL of streptavidin beads were added, in a total volume of 200 μL. Samples were washed six times, eluted in fresh 100 mM DTT, and further purified using the RNA Clean & Concentrator-5 kit (Zymoresearch). Libraries for RNA-seq were constructed using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®.
Biotinylated 4sU-RNA was recovered using the μMacs Streptavidin Kit (Miltenyi). Per microgram of recovered biotinylated 4sU-RNA, 0.5 μL of streptavidin beads were added, in a total volume of 200 μL. Samples were washed six times, eluted in fresh 100 mM DTT, and further purified using the RNA Clean & Concentrator-5 kit (Zymoresearch). Libraries for RNA-seq were constructed using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®.