title: GSM1976593: IgG_iCLIP_zf_ZGA_rep3; Danio rerio; OTHER
library_construction_protocol: Zebrafish embryos were lysed and lysates treated with RNaseI to obtain optimal length of RNA fragments for cDNA library preparation. hnRNP A1-RNA complexes were immunopurified using mouse anti-hnRNP A1 antibody and protein G-dynabeads (Invitrogen). Anti-IgG antibody (Sigma) was used for negative control immunopurifications. After RNA dephosphorylation, 3' end linker ligation was performed. Protein-RNA complexes were resolved on SDS-PAGE and transferred to the nitrocellulose membrane. Region of the membrane above the predicted size of hnRNP A1 was cut and RNA extracted using proteise K treatment. Subsequently, RNA was reverse transcribed using RT primers harboring a barcode. cDNA was separated into three size selected fractions using 6% TBE-Urea gel (Life Technologies) and circularized by ssDNA circligase. Specific oligo was annealed for BamHI restriction site formation. cDNA was linearized by BamHI digestion. Final cDNA libraries were generated by PCR amplification.. 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
attributes.tag: GEO Accession
attributes.value: GSM1976593