PC3_etoposide_1
The construction of RPF libraries was done as previously described (M. Van de Wetering, Cell ,2015), with no addition of harringtonine treatment. Total RNA was isolated using Trizol reagent (Invitrogen), according to manufacturers instructions and rapid gel extraction was used followed by overnight precipitation at 20 C for RNA extraction from the polyacrylamide gels. rRNA depletion was not performed and rRNA was removed during the data processing. The pooled library was analysed on a 2100 Bioanalyser using a 7500 chip (Agilent, Santa Clara, CA), quantified by qPCR (Cat.no. 07960140001, Roche, Switzerland) and subsequently diluted to 10nM. Next, the library was loaded with 1.5pM on a NextSeq550Dx system (RUO mode) and sequenced with 75-base single-read using a 75 Cycles High Output Kit v2.5 (Illumina Inc., San Diego).