GSM9415221: TT-seq, SSRP1-FKBP-E14, DMSO, clone1; Mus musculus; OTHER
Fifty million cells were labelled with 500uM 4-thiouridine (4sU) for 5 min and lysed in Trizol. For reference normalization, Drosophila S2 cells were labelled with 500uM 4sU for 5min and lysed in Trizol. Drosophila lysate was added in a ratio of 1:3 (drosophila:mouse). Total RNA was isolated by phenol-chloroform extraction, DNAse treated and sonicated (Covaris ME220). 4sU-labelled transcripts were biotinylated with EZ-link biotin-HPDP (ThermoFisher Scientific), isolated using Streptavidin beads (Miltenyi) and purified with the RNeasy MinElute Cleanup Kit (Qiagen 74204). Libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit (E7760).