PIP-seq is reproducible and captures known RBP–RNA interactions. (A) Correlation in read counts between two formaldehyde-cross-linked ssRNase-treated PIP-seq replicates (footprint sample on left, RNase digestion control on right). (B) As (A), but for formaldehyde-cross-linked dsRNase-treated replicates. (C) Overlap in PPS calls between formaldehyde-cross-linked ssRNase-treated (top, blue), and formaldehyde-cross-linked dsRNase-treated (bottom, green) PIP-seq replicates. (D) Overlap between PPSs identified from three formaldehyde-treated PIP-seq samples and various CLIP datasets. Values are shown as log2 enrichment over shuffled background distributions. *** denotes P < 2.2 × 10-16 (chi-squared test). (E) Overlap between formaldehyde-cross-linked PPSs from HeLa cells and 40-nucleotide T > C transversion event-containing loci from the gPAR-CLIP dataset generated from HEK293T cells (T > C transversion events less than 40 bp apart were merged to generate a dataset comparable to PPSs). (F) Number of T > C transversion events per PPS identified by formaldehyde cross-linking (purple) versus shuffled regions (gray). Values for the number of events per shuffled region are the average from ten random shuffles. bp, base pair; dsRNase, double-stranded RNase; PIP-seq, protein interaction profile sequencing; PPS, protein-protected site; ssRNase, single-stranded RNase.