eCLIP-seq of TRAP1 in PEA1 cells
Enhanced crosslinking and immunoprecipitation sequencing (eCLIP) libraries were constructed using the RBP-eCLIP kit (ECLIPSEbio) according to the manufacturers instructions. Briefly, PEA1 cells were UV cross-linked on ice at 0.40 J/cm2 with UV light at 254 nM, followed by cell lysis and lysate treatment with RNAse I to shear RNA. TRAP1 protein-RNA complexes were immunoprecipitated. Stringent washes were performed, during which RNA was dephosphorylated with FastAP and T4 PNK. Subsequently, a 3 RNA adapter was ligated onto the RNA with T4 RNA ligase. Protein-RNA complexes were run on an SDS-PAGE gel, transferred to nitrocellulose membranes, and RNA was isolated off the membrane identically to standard iCLIP. After precipitation, RNA was reverse transcribed with AffinityScript (Agilent), free primer was removed (ExoSap-IT, Affymetrix), and a 3 DNA adapter was ligated onto the cDNA product with T4 RNA ligase (NEB). Libraries were then amplified with Q5 PCR mix (NEB).
SUB15178795
PEA1_IP3_R1_001