Enhanced cross-linked immunoprecipitation follow by sequencing (eCLIP-seq) of PCBP1 in embryonic mouse heart

PCBP1 is an RNA-binding protein abundantly expressed in the embryonic heart. We sought to determine the RNA interaction of Pcbp1 in embryonic heart. Overall design: Embryonic day (E) 16.5 C57BL/6N hearts ventricles were snap-frozen follow by cryogrinded, UV crosslinking, and sonication. 20 x E16.5 hearts were pool per biological replicate (40 hearts total, n=2). Immunoprecipiation was performed by a pre-validated PCBP1 antibody (Abcam cat #ab168377). 2% of the sample was taken as the paired input sample. IP and input samples were cut from the membrane at the relative band size of 40kDa to 75kDA above that band size. RNA adapter ligation, reverse transcription, DNA adapater ligation and PCR amplification were performed.