GSM5013970: chase_4sU_merged.4-OHT.minus/plus_strand; Homo sapiens; RNA-Seq
SH-EP-MYCNER cell lysates were spiked with 4sU-treated mouse T-cell lysates for the no chase 4sU. Total RNA was isolated using QIAzol and purified with miRNeasy columns and biotinylated. This was followed by  4sU-RNA pulldown pulldown using streptavidin-MyOne beads, followed by purification with RNA minElute columns (Qiagen) 4sU-RNA was used for library preparation with NEBNext rRNA Depletion Kit (Human/Mouse/Rat) and NEBNext Ultra II Directional RNA Library Prep kit. The libraries were amplified with 10-14 PCR cycles depending on the input RNA. The concentration and size distribution of the libraries were determined on a Fragment Analyzer using the NGS Fragment High Sensitivity Analysis Kit (1-6,000 bp; Agilent Technologies). The libraries were sequenced on the NextSeq500 Illumina platform for 75 cycles. Base calling was performed using Illumina's BaseSpace platform.