RNA immunoprecipitation (RIP) of KHSRP from pancreatic cancer cell extracts cultured on fibronectin

KHSRP-bound transcripts were identified by RNA immunoprecipitation and sequencing (RIP-Seq) and bioinformatics analysis. A pancreatic cancer cell line S2-013 was cultured on fibronectin. We performed RIP of KHSRP using anti-KHSRP antibody from the fibronectin-stimulated S2-013 extracts, and identified the associated RNAs using a next generation sequencer. S2-013 cells were seeded on fibronectin and incubated for 5.5 h. After washing twice with PBS, cells were lysed in NP2 buffer. Lysates were immunoprecipitated with anti-KHSRP antibody or with normal rabbit IgG and Dynabeads Protein G. The immunoprecipitation products were processed by Hokkaido System Science (Sapporo, Japan) using an Illumina/Solexa instrument following standard procedures.