Design of protein occupancy profiling experiments and differential occupancy analysis. (A) Schematic representation of the experimental approach of protein occupancy profiling on RNA. Photoreactive ribonucleosides are incorporated into newly synthesized RNA. Protein-RNA complexes are crosslinked with low-energy UV light (365 nm). Crosslinked polyadenylated transcripts are captured by oligo(dT) affinity purification and RNAse I treated. Protein protected RNA fragments are subsequently subjected to small RNA cloning and Illumina sequencing. (B) Overview of the differential T-C transition normalization and statistical testing scheme. For each annotated transcript that passed filtering criteria, initial normalization shifts T-C transition counts for all replicates of the two conditions to the same distributions, thereby removing differences that might arise from variations in sequencing depth or mRNA expression levels of that particular gene (indicated in light blue). Subsequently, a negative binomial testing scheme is used to identify positions with significantly increased or decreased protein occupancy. CDS, coding sequence.