Figure 1

Processing of PAR-CLIP RNA samples for sequencing. (a) Outline of experiments. PAR-CLIP of endogenous PARP1 was performed on HeLa cells using 4-thiouridine (4SU). (b) PAR-CLIP samples run on an SDS PAGE gel were imaged with Typhoon. (c) Protein-bound samples were transferred onto a nitrocellulose membrane and exposed to a phosphoimager screen and imaged using the Typhoon. (d) The same membrane in a was probed for the presence of PARP1-bound RNAs using an antibody to PARP1. Red arrows (b–d) indicate PARP1 bound-RNA—one at ~140 kDa and a shorter fragment at ~100 kDa. As the antibody recognizes the larger fragment, we considered this as the full-length protein. The lower band could be a proteolytic fragment as determined by mass spectrometry, lacking the N terminus, and rendering it undetectable by the antibody raised against the N-terminal domain of the protein. (e) Processing of PARP1-bound RNAs for sequencing. A representative denaturing (8 M urea) polyacrylamide gel showing the different steps of adapter ligation to RNA samples. PARP1-bound RNAs were eluted from membrane in c, deproteinized, ligated to 3′ and 5′ adapters (lane 4). Lanes 1 and 6 are control 19-mer and 24-mer labeled RNAs, ligated to 3′ adapter. These ligated 3′ adapter control RNAs were further ligated to 5′ adapters (lanes 2 and 7). The green arrow indicates unligated 19-mer and 24-mer RNAs (Lanes 1 and 6, respectively); the blue arrow indicates 3′-adapter ligated control RNAs; the red arrow indicates 3′ adapter and 5′-adapter ligated control RNAs. These controls were used to test the ligation efficiency of our samples. The black arrow (lane 4) indicates 3′ and 5′ adapter ligated PARP1-bound RNA samples. (f) Adapter-ligated samples were subjected to limited PCR amplification. Lanes 1 and 2 show 3′ and 5′ ligated 19-mer and 24-mer control RNAs converted to cDNA and PCR-amplified. Lanes 3 and 4 are the PARP1-bound RNAs subjected to cDNA conversion and PCR amplification. The black arrow shows the PCR products used for sequencing.