RNA immunoprecipitation sequencing (RIP-seq) of BUD31 in ovarian cancer cells

Mis-regulation of splicing factors are thought to activate cancer specific splicing programs that contribute to cancer development and progression. However, it remains a major challenge to identify the key splicing variants caused by aberrant expression of splicing factors in cancer. Here we report that splicing factor BUD31 is commonly overexpressed in high-grade serous ovarian carcinoma (HGSOC) and high level of BUD31 is associated with poor prognosis. RNA-seq analysis reveals that BUD31 inhibition predominantly results in alternation of exon skipping and intron retention. RNA immunoprecipitation sequencing (RIP-seq) was used to analysis the interaction between BUD31 and the target RNA. Our data indicate that BUD31 is a critical oncogenic splicing factor in ovarian cancer and might act as a potential therapeutic target. Overall design: RNA immunoprecipitation sequencing (RIP-seq) was performed in HEY cells with BUD31 antibody . HEY cells were collected for the RIP assay, which was performed using the EZ-Nuclear RIP Kit (17-701, Merck Millipore) following the manufacturer's instructions. In brief, cells were collected and lysed in RIP lysis buffer. Lysates were incubated with magnetic beads coated with anti-BUD31 antibody (Proteintech, 11798-1-AP) at 4 °C overnight. The beads combined with immunocomplexes were washed with RIP wash buffer for 6 times and digested by protease K. RNA was extracted with Phenol/Chloroform/Isoamyl Alcohol (125:24:1 Mixture).