Identification of transcriptome-wide mRNA targets of the ELAVL3 protein
PAR-CLIP from SHSY5Y cells over-expressing FLAG-HA-tagged ELAVL3 protein was performed as described previously (Hafner et al. 2010). Prior to conducting the PAR-CLIP, the cells were differentiated adding (1) 10 µM retinoic acid and growing for 5 days, (2) BDNF (50 µM) and growing for an additional 5 days. On day 4 of BDNF treatment, the growth medium of SHSY5Y cells was supplemented with 100 µM 4-thiouridine (4SU) for 24 hours prior to crosslinking. After decanting the growth medium, cells were irradiated uncovered with 0.15 J/cm2 of 365 nm UV light in a Stratalinker 2400. A 5 ml pellet was processed to whole lysate. FLAG-HA-tagged-ELAVL3 was immunoprecipitated with anti-FLAG antibody conjugated to protein G Dynabeads. The radiolabeled band corresponding to ~50 kDa ELAVL3-RNA complex was excised, the associated RNA isolated by phenol-chloroform extraction following proteinase K treatment, converted to a cDNA library and Illumina sequenced at the Rockefeller University Genomics Center. Note: (1) Submitted file is the raw sequencing file without any processing. (2) For information regarding how to process see PMID: 24860013, for a dataset collected and processed similarly.
Rockefeller University
ELAVL3_SHSY5Y_differentiation