GSM2508007: PARP1_PAR-CLiP; Homo sapiens; OTHER
PAR-CLIP was performed as previously described (Spitzer et al, 2014) with some modifications. Briefly, 10 ml of packed cell pellet - UV-treated cells, were lysed with 3 volumes of 1× NP40 lysis buffer on ice for 5 mins. Cells were pelleted by centrifugation at 18,000g for 15 min using an Eppendorf 5810R centrifuge 5810R with an A-4-81swing bucket rotor.  The supernatant was filtered using a 5 µm syringe filter (Nalgene) to remove cellular debris. The filtrate was then partially treated with RNase T1 (Roche) to a final concentration of 1 U/µl for 15 min. The supernatant was then incubated with 600 μl of protein A dynabeads (Invitrogen) bound to 15 μg of anti-PARP1 antibody (Active Motif) or control IgG antibody for 2 h. The beads were washed 3 times and the immunoprecipitated RNA was digested again with RNase T1 to a final concentration of 63 U/µl for 15 min.  After dephosphorylation, the RNA segments crosslinked to PARP1 were 5’-radiolabeled using γ-32P-ATP and T4 polynucleotide kinase (Promega) in one original bead volume. After several washes, each CLIP sample (on the beads) was then treated with 5U of DNase1 (NEB) for every 100 μl of bead volume for 15 min at 37°C. DNase1 was inactivated by adding 5 mM EDTA and heated at 65°C for 10min. Samples were then resuspended in SDS-PAGE loading buffer, incubated at 95°C for 5 min to denature and release the PARP1-RNA cross-links. The samples were then separated on 4 –12 % NuPAGE gels (Invitrogen) and transferred onto nitrocellulose membranes (1/10th of the sample was used for immunoblotting and the rest of the sample was used for autoradiography). The gel containing 1/10th of the sample and the membrane containing 9/10th of the sample were exposed to a phosphorimager screen overnight and visualized by scanning on a Typhoon FLA 9500. PARP1–RNA complexes were cut from the membrane, treated with proteinase K (Roche) followed by Phenol/Chloroform/IAA extractions and ethanol precipitation. The recovered RNA further was used for cDNA library preparation. For this purpose we used NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 1). Library preparation including 3’ and 5’ SR Adaptor ligations, reverse transcription and PCR amplification, were performed per manufacturer’s protocol. To remove adaptor-only ligation products, after every step of the protocol (3’adapter ligation and 5’adapter ligation) samples were purified using 15% acrylamide-8M Urea gels. Lastly after limited PCR amplification PCR products were size-selected on a 3.5% NuSieve (Lonza) low-melting point agarose gel. Expectant PCR products were eluted using the ‘crush and soak’ method, followed by purification using a Qiagen min-elute PCR column. Samples were then first cloned into the Topo TA vector for pilot analysis and then sequenced using Illumina sequencing.