Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions
Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions
Mouse brain tissue (Nova iCLIP) or COS-7 cells grown in a tissue culture plate (mutant NSUN2 iCLIP). Tissue was dissociated in ice-cold PBS buffer (Nova iCLIP) or medium was replaced (mutant NSUN2 iCLIP). Material was then subjected to UV-C irradiation. Cells were then precipitated by centrifugation for 1 min at 14000 rpm and snap-frozen on dry ice. iCLIP protocol was followed to isolate RNA bound to Nova protein or mutant NSUN2 protein (C271A) and amplify cDNA library as described in the manuscript. Specific barcodes were used for each RT reaction, and are specified below for each sequencing run. Random barcodes were also incoperated into cDNAs to distinguish between PCR duplication of cDNA products. The final products were obtained by PCR with Illumina pair-end sequencing primers: 5-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3; 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3
Experimental Factor: IMMUNOPRECIPITATE
anti_Nova_antibody