GSM5737633: IGF2BP2 IP 1; Homo sapiens; RIP-Seq
The immunoprecipitates were further incubated with protein A /G Dynabeads for 2h at 4 °C. After applying the magnet and removing the supernatant, we sequentially washed the beads 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, 20 mM EGTA and 0.5% NP-40) twice. The beads were then resuspended in Elution buffer (50 nM Tris 8.0, 10 mM EDTA and 1% SDS) and the suspension was incubated for 20 min in a heat block at 70°C to release the immunoprecipitated RBP with crosslinked RNA before vortex. The magnetic beads were removed from the separator and the supernatant was transferred to a clean 1.5 ml microfuge tube. Proteinase K (Roche) was added to the 1% input (without immunoprecipitated）and the immunoprecipitated RBP with crosslinked RNA to a final concentration of 1.2 mg/ml, and the mixture was incubated for 120 min at 55°C. The RNA was purified with the Trizol reagent (Life technologies, USA).  cDNA libraries were prepared with the KAPA RNA Hyper Prep Kit (KK8541, KAPA, Boston, USA) according to the manufacturer's protocol. We prepared high-throughput sequencing libraries following the manufacturer's instructions for the Illumina NovaSeq 6000 system with 150 nt paired-end sequencing.