E-MTAB-3534
iCLIP analysis of RNA bound by FUS in human brain
RNA abundance decreases linearly from the 5' to 3' end of long introns to create “saw-tooth” patterns, and these can be used to infer locations of major splicing events. FUS binds across entire pre-mRNAs with limited sequence specificity, permitting examination of these saw-tooth patterns. We used FUS iCLIP from the human brain to identify deviations from the expected linear decrease of reads across long introns that correspond to recursive splicing events.  iCLIP protocol was followed to isolate RNA bound to FUS protein and amplify cDNA library as described in the manuscript. Specific barcodes were used for each RT reaction, and are specified in the library construction protocol for each sequencing run. Random barcodes were also incorporated into cDNAs to distinguish between PCR duplication of cDNA products. The final products were obtained by PCR with Illumina paired-end sequencing primers: 5-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3; 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3
RNA abundance decreases linearly from the 5' to 3' end of long introns to create “saw-tooth” patterns, and these can be used to infer locations of major splicing events. FUS binds across entire pre-mRNAs with limited sequence specificity, permitting examination of these saw-tooth patterns. We used FUS iCLIP from the human brain to identify deviations from the expected linear decrease of reads across long introns that correspond to recursive splicing events.  iCLIP protocol was followed to isolate RNA bound to FUS protein and amplify cDNA library as described in the manuscript. Specific barcodes were used for each RT reaction, and are specified in the library construction protocol for each sequencing run. Random barcodes were also incorporated into cDNAs to distinguish between PCR duplication of cDNA products. The final products were obtained by PCR with Illumina paired-end sequencing primers: 5-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3; 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3