GSM8888765: input; Mus musculus; RIP-Seq
First, harvest cells and wash them with 1xPBS. Then, resuspend the cells in RIP buffer and incubate on ice for 20 min. Next, centrifuge the cell suspension at 15,000 x g for 15 min at 4 °C to clarify the lysate. After that, wash the beads three times with RIP buffer, add the cell supernatant to the beads, and incubate with rotation at 4 °C for 2 h. Subsequently, remove the beads and incubate the supernatant with the corresponding antibody for 2 h. Finally, wash the sample three times and extract RNA for detection. After immunoprecipitation, the enriched DNA was purified using the QIAquick PCR purification kit (Qiagen). DNA was then end-repaired, A-tailed, and ligated to indexed adapters. The ligated DNA was amplified by PCR, and the resulting libraries were purified using the QIAquick PCR purification kit. Library quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by qPCR. The libraries were then sequenced on the Illumina HiSeq 3000 platform, generating 150 bp paired-end reads.