GSM9270307: rip_ctrl; Mus musculus; RIP-Seq
Mouse livers were collected after euthanasia and extracted using Tissue Lyser (Qiagen) in Nonidet P-40 lysis buffer supplemented with 1 U/μl RNase Inhibitor (FEREO0381PM, Fisher Scientific) and extracted for 10 min. The lysate was centrifuged for 10 min at 16,800g, and supernatant was transferred to a fresh 1.5-ml tube. Total protein was measured by Bradford assay and 2 mg of cytoplasmic lysate protein was subjected to immunoprecipitation. Lysates were incubated with Flag antibody-conjugated beads for 6-8 hours at 4°C. The beads were subsequently washed three times with 1 mL of Nonidet P-40 lysis buffer supplemented with 1 U/μl RNase Inhibitor. The RNA and protein on beads were isolated using TRIzol according to the manufacturer's instructions. The RNA fraction was subjected to qPCR analysis and RNA sequencing (RNA-seq). With the RNA from RIP, cDNA synthesis and library preparation were carried out using the SMART-Seq mRNA LP kit (Takara, 634768).