GSM6322747: WT_plusIAA_R3; Homo sapiens; OTHER
U2OS cell lysates were spiked with 4sU-treated mouse T-cell lysates. Total RNA was isolated using QIAzol, purified with the miRNeasy mini kit, precipitated to reduce volume and biotinylated. This was followed by 4sU-RNA pulldown using MyOne streptavidin beads, followed by purification with the RNeasy MinElute cleanup kit 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 11 PCR cycles. 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.