title: GSM6564532: RPS27_IP-2; Homo sapiens; RIP-Seq
library_name: GSM6564532
library_construction_protocol: The immunuprecipitates were further incubated with protein A/G Dynabeads (Thermo Scientific) for 2h at 4 °C. After applying to magnet and removing the supernatants, the beads were sequentially washed with lysis buffer, high-salt buffer (250 mM Tris 7.4, 750 mM NaCl, 10 mM EDTA, 0.1% SDS, 0.5% NP-40 and 0.5 deoxycholate), and PNK buffer (50 mM Tris 7.4, 20 mM EGTA and 0.5% NP-40) for two times, respectively. Resuspend the beads in of Elution buffer (50 nM Tris 8.0, 10 mM EDTA and 1% SDS). Incubate the suspension for 20 min in a heat block at 70°C to release the immunoprecipitated RBP with crosslinked RNA and vortex. Remove the magnetic beads on the separator and transfer the supernatant to a clean 1.5 ml microfuge tube. Add Proteinase K (BBI) into the 1% input（without immunoprecipitated）and immunoprecipitated RBP with crosslinked RNA, with final concentration of 1.2 mg/ml. Incubate for 120 min at 55°C. The RNA was purified with Trizol reagent (Life technologies). cDNA libraries were prepared with the KAPA RNA Hyper Prep Kit (KAPA, KK8541) according the manufacturer's procedure. For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina NovaSeq system for 150 nt paired-end sequencing.