Nascent-seq of HeLa cells after rapid depletion of SRSF5 using hGRAD for 2h, 8h and 16h
We used HeLa cells expressing GFP-tagged SRSF5 and the rapid depletion plasmid hGRAD from genomic loci. SRSF5 depletion was started after addition of Doxycyclin and was allowed for 2h, 8h and 16h. One hour before harvesting, cells were incubated with 4-sU for exactly 1 hour to label newly transcribed RNAs. 4-sU-labelled RNAs were biotinylated and purified over streptavidin beads and transformed into an RNA-seq library. We compared differential expression and intron retention between T0 and the other time points. Overall design: We sequenced 2 biological replicates for all four timepoints (T0, T2, T8 and T16) with 150-bp paired end reads and 100 Million reads per replicate.