Crosslinking Immunoprecipitation for TDP-43 and CELF2

The iCLIP protocol was performed as described previously10 (link), with the following modifications. SH-SY5Y neuroblastoma or H9 human embryonic stem cells were irradiate once with 150 mJ/cm² in a Stratlinker 2400 at 254 nm, and brain tissue was dissociated in cold PBS and the suspension was crosslinked four times with 100 mJ/cm². TDP-43 was immunoprecipitated with protein A Dynabeads (Invitrogen) conjugated to rabbit-anti TDP-43 (Proteintech, 10782-2-AP). For iCLIP of CELF2, protein G Dynabeads conjugated to mouse anti-CELF2 (Sigma, C9367) were used. In both cases, the region corresponding to 55–100kDa complexes was excised from the membrane to isolate the RNA. High-throughput sequencing using Illumina GA2 was done using 54 or 72 cycles (Supplementary Table S1). The barcode sequences corresponding to the individual experiment were as described (Supplementary table 1). The random barcodes were registered and the barcodes were removed before mapping the sequences to the human genome sequence (version GRCh37/hg19) allowing one mismatch using Bowtie version 0.10.1 (command line: -a -m 1 -v 1).