GSM4182482: mSRSF7_piCLIP_mm9_poly_IP_GGCG_Rep2; Mus musculus; RIP-Seq
Cells were lysed and treated with RNase I. GFP-tagged proteins were immunoprecipitated with Gamma binding beads coupled to goat anti-EGFP antibody. Endogenous SRSF7 was immunoprecipitated with Gamma binding beads coupled to rabbit anti-SRSF7 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 containing a barcode sequence. cDNA was size selected by denaturing gel electrophoresis and circularized by ssDNA circligase. A specific oligonucleotide was annealed to create a restriction site for BamHI and cDNA was linearized by digest with BamHI. Linearized cDNA was PCR amplifiied with Solexa sequencing primers to create the final libraries. For more details see: Huppertz et al., (2014): iCLIP: protein-RNA interactions at nucleotide resolution. Methods. PMID:24184352
GEO Accession
GSM4182482