Anti-Ybx1 RIP-seq to identify target mRNAs of Ybx1 in E13.5 DRG [RIP-seq]
In order to identify the target mRNAs of the m5C reader protein Ybx1, we carried out anti-Ybx1 RNA Immunoprecipitation(RIP) followed by RNA-sequencing to find out mRNAs that can directly bind by Ybx1. Using EZ-Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore), RNA from E13.5 wildtype mouse DRGs was pulled down by rabbit monoclonal anti-Ybx1 (abcam) and sequenced on IIIumina HiSeq3000 platform. The filtered reads were mapped to the mouse reference genome (GRCm39) using BWA mem (v 0.7.12). After peak calling using MACS2 (version 2.1.0) software, the distribution of chromosome distribution, peak width, fold enrichment, significant level and peak summit number per peak were all displayed. Fold enrichment greater than 2 was considered to be enriched. Finally, 2 biological repilicates of anti-Ybx1 RIP-seq identified 2487 transcripts. This study provides a gene list which shows mRNA binding with Ybx1 in mouse DRGs. Overall design: Anti-Ybx1 RNA immunoprecipitation was performed after lysing dissociated DRGs from E13.5 wildtype mice. Two biological replicates were used, each containing input and IP samples.