GSM7287618: HeLa, DMSO, 4sU 120 min, rep1; Homo sapiens; RNA-Seq
RNA was extracted with the GeneMatrix universal RNA purification kit (Roboklon). Ribosomal RNA was depleted with the NEBNext rRNA Depletion Kit (NEB E6350L). To enhance the efficiency of 4sU-to-C conversion, RNA was fragmented to an average length of ~200 nt by alkaline hydrolysis at pH 10.0 for 7minutes at 95 °C. Conversion of 4sU to C was induced by treatment with 10 mM NaIO4 in 600 mM NH4Cl (pH 8.5) for 15 min at 40 °C as described previously for ribosome-protected fragments (PMID: 34480152). Sequencing libraries were prepared with the NEBnext Ultra II Directional RNA Library Prep Kit and sequenced single-end on a NextSeq550 device (Illumina). The 30 minute and 2 hour time-point libraries were sequenced to a length of 150 and 80 nt, respectively, to account for the lower 4sU incorporation rate after 30 min of 4sU treatment.