GSM7158903: USP39 iCLIP2 high Replicate 1; Homo sapiens; RIP-Seq
Cells were lysed and treated with RNase I. USP39 was immunoprecipitated with Gamma binding beads coupled to anti-USP39 antibody. After stringent washing, RNA was dephosphorylated and ligated to a 3' linker. Protein-RNA complexes were resolved by denaturing gel electrophoresis and transfered to nitrocellulose membrane. Two different membrane regions above the expected size of the protein (Low and High) were cut and RNA was released by Proteinase K treatment. RNA was reverse transcribed using reverse primer and a second adapter containing a barcode was ligated to the cDNAs. The cDNA was pre-amplified for 6 cycles using short Solexa P3 and P7 primers. After a size selection on beads a second PCR amplification was performed with the normal P3 and P7 primers to generate the final iCLIP2 libraries. iCLIP2