RNA immunoprecipitation-PCR (RIP-PCR) and in vivo crosslinking followed by immunoprecipitation (CLIP)

MCF7 cells cultured in 10 cm dishes were harvested, and RIP assays were carried out using SRSF1 (sc-33,652) antibodies and a Magna RIP kit (Millipore). The RIP assay was performed based on the instructions of the Magna RIP kit. RNA was extracted from the immunoprecipitate and reverse transcribed into cDNA for further RT-PCR detection. Primer sequences are listed in Supplementary Table 4.

An in vivo CLIP assay of RNA bound directly to SRSF1 was carried out as previously described with mild modifications [38]. Briefly, MCF7 cells were harvested, and ultraviolet cross-linking was conducted followed by immunoprecipitation using SRSF1 antibodies and a Magna RIP kit. Cell extracts were incubated with antibody-coated magnetic beads overnight at 4 °C followed by washing with wash buffer 3 times, and 100 μl of wash buffer was added. Then, the cells were incubated with RNaseT1 (100 U/μl) in 22 °C water for 1 h, followed by immediate incubation in an ice bath for 5 min. Beads were washed 3 times with wash buffer and then subjected to proteinase K digestion, RNA extraction and reverse transcription using random primers. RT-PCR assays were carried out with specifically designed primers to amplify skipped cassette exons and flanking exons. Primer sequences are listed in Supplementary Table 4.