GSM2631832: iCLIP in Cntl cells-rep2; Homo sapiens; OTHER
HeLa cells were irradiated with UV light at 300 mJ/cm2.  After cell lysis, RNAs were partially fragmented using 0.5 U/ml of RNase I, followed by immunoprecipitaions with or without (no antibody control) 10 μg of the ALYREF antibody immobilized on protein A Dynabeads (Life Technologies).  After extensive washing, immunoprecipitated RNAs were ligated at the 3’ ends to a RNA adapter and radioactively labeled by T4 polynucleotide kinase (Fermentas).  The protein-RNA complexes were then transferred to a nitrocellulose membrane.  Then, fragmented RNAs were purified from nitrocellulose membrane by PK treatment and phenol / chloroform extranction. For iCLIP cDNA library preparation, fragmented RNAs, which were ligated with a RNA adapter (3' linker)  at the 3’ ends , reverse-transcribed with a primer containing a barcode (iCLIP RT primer).  The resulting cDNAs were purified by PAGE, circularized by single-stranded DNA ligase (Epicentre), linearized by restriction enzyme cleavage, and amplified and add P5/P3 adaptors by PCRs with (P3 and P5 solexa primer). iCLIP-seq
GEO Accession
GSM2631832