title: GSM5195718: 4SU_shMYC_Dox_pulse_rep1; Mus musculus; OTHER
library_construction_protocol: For 4SU-seq in vitro experiments, KPC cells were labelled with 200 µM 4-thiouridine for 20 min, lysed with QIAzol reagent and spiked with 4sU-labelled U2OS cell-lysates. Total RNA was extracted with the miRNeasy kit (Qiagen) including on-column DNase digestion and quantified. The isolated RNA was biotinylated with EZ-Link Biotin-HPDP (Pierce) for 2 hours followed by purification of biotinylated RNA via chloroform-isoamylalcohol extration. Biotinylated RNA was pulled down using Dynabeads MyOne Streptavidin T1 beads (Invitrogen) . Following washing, 4sU-labelled RNA was eluted from the beads, purified with the RNeasy MinElute kit (Qiagen) and quantified with the RiboGreen Assay (Invitrogen). About 10 ng of RNA was used as input material for library preparation. First, the rRNA was depleted using the NEBNext rRNA Depletion Kit (NEB), followed by cDNA preparation and library preparation using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB). The cDNA libraries were amplified with about 12 PCR cycles and quality and quantity and the size of the cDNA libraries were determined with the Fragment Analyzer (Thermo Fisher Scientific, previously Advanced Analytical Technologies). Libraries were sequenced on a NextSeq500 System (Illumina).