title: GSM2806530: K8 CLIP-seq  in BJAB cells; Homo sapiens; RIP-Seq
library_construction_protocol: Turbo DNase was added into the cell lysate. Lysate was incubated on ice for 30 min and spun downed at 4°C and 20,000 g for 10 min to clear the lysate. Antibody of interest and 50μl washed protein A Dynabeads was added per 500μl lysates. Samples were rotated for 2 h at 4°C. Discarded the supernatant and washed the beads twice with 900 μl high-salt buffer. Then washed twice with 900 μl wash buffer with different mount of Rnase A (high-RNase concentration: 2μg/ml, moderate-RNase concentration: 1μg/ml and low-RNase concentration: 0.2μg/ml). Beads were washed with 900 μl RNase-free wash buffer twice. Then RNA 3’ ends were dephosphorylated. Linkers were ligated to 3’ ends of RNAs, 32P-γ-ATP labeled linker was ligated to the 5 ’ends of RNAs. The protein-RNA complexes were resolved on a 4-12% NuPAGE Bis-Tris gel and transferred to a nitrocellulose membrane. K8-RNAs complexes were isolated and subjected to proteinase K digestion and RNA purification by TRIzol. RNAs were subjected to RT-PCR according to TruSeq™ RNA and DNA Sample Prep Kits.
attributes.tag: GEO Accession
attributes.value: GSM2806530