GSM8058154: Arid5a iCLIP2 Replicate 1; Mus musculus; RIP-Seq
Cells were lysed and treated with RNase I. Arid5a was immunoprecipitated with Gamma binding beads coupled to anti-GFP 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. A membrane region above the expected size of the protein was 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