RNA Immunoprecipitation, High-Throughput Sequencing (RIP-seq), and Data Analysis

The cell sample was suspended in a cell culture dish with precooled PBS, and the dish was irradiated with a UV cross-linker. Collected cells were added to the lysate at a ratio of 1:10 to lyse and digest the DNA. The lysate was incubated with the RIP antibodies overnight at 4°C for immunoprecipitation. The magnetic beads were resuspended with the antigen-antibody complex solution for 1 h, and the magnetic beads resuspended in MNase were placed on a hot mixer for 10 min and eluted. After removal of MNase, the sample was dephosphorylated with FastAP enzyme, treated with T4 PNK enzyme, and then treated with proteinase K to digest protein, and the RNA was extracted. The Illumina ScriptSeqTM v2 RNA-Seq Library Preparation Kit (Li et al., 2017; Song et al., 2018) was used for the library construction and sequencing. The quality of the RIP library was judged according to the mapping result. Quality-qualified data were entered for the downstream analysis to obtain valid reads for the genomic location distribution, peak calling, and motif analysis, which revealed the type and pattern of mRNA and ncRNA that Prdx1 binds at the genome-wide level.