GSM1411612: Coilin_iCLIP_Mm_rep4; Mus musculus; RIP-Seq
Cells were lysed and treated with RNase I. Coilin-GFP was immunoprecipitated with Gamma binding beads coupled to goat anti-EGFP antibody. After stringent washing, RNA was dephosphorylated and ligated to 3' linker. Protein-RNA complexes were resolved on denaturing gel electrophoresis and transfered to nitrocellulose membrane. A membrane region above the protein expected size was cut and RNA was released by Proteinase K treatment. RNA was reverse transcribed using reverse primer containing a barcode sequence. cDNA was separated into 3 size fractions by denaturing gel electrophoresis and circularized by ssDNA circligase. Specific oligo was annealed to create restriction site and cDNA was linearized by digest with BamHI. Linearized cDNA was PCR amplifiied to create final library. For more details see: König et al. (2010): iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol PMID:20601959
GEO Accession
GSM1411612