Project description:RNA-binding proteins (RBPs) and non-coding RNAs orchestrate post-transcriptional processes through the recognition of specific sites on targeted transcripts. Thus, understanding the connection between binding to specific sites and active regulation of the whole transcript is essential. Many immunoprecipitation techniques have been developed that identify either whole transcripts or binding sites of RBPs on each transcript using cell lysates. However, none of these methods simultaneously measures the strength of each binding site, and quantifies binding to whole transcripts. In this study, we compare current procedures and present Digestion Optimized (DO)-RIP-seq, a simple method that locates and quantifies RBP binding sites using a continuous metric. We have used the RBP HuR/ELAVL1 to demonstrate that DO-RIP-seq can quantify HuR binding sites with high coverage across the entire human transcriptome, thereby generating metrics of relative RNA binding strength. We demonstrate that this quantitative enrichment of binding sites is proportional to the relative in vitro binding strength for these sites. Also, we used DO-RIP-seq to quantify and compare HuR's binding to whole transcripts, thus allowing for seamless integration of binding site data with whole transcript measurements. Finally, we demonstrate that DO-RIP-seq is useful for identifying functional mRNA target sets, and binding sites where combinatorial interactions between HuR and AGO-microRNAs regulate the fate of the transcripts. Our data indicate that DO-RIP-seq will be useful for quantifying RBP binding events that regulate dynamic biological processes.
Project description:The analysis of the RNA-binding protein immunoprecipitation sequencing (RIP-seq) experiment is performed to identified YAP-specific binding lncRNAs.
Project description:We identified the mRNA targets of the insulin-like growth factor-2 (IGF2) mRNA-binding proteins 1, 2, and 3 (IGF2BP1/2/3) by RNA immunoprecipitation and sequencing (RIP-seq). HEK293T cells transfected with Flag-tagged IGF2BP1/2/3 plasmids were expanded and UV-crosslinked before harvest. We performed RIP of individual IGF2BP using anti-Flag antibody from nuclear extractions, and identified the associated mRNAs by next generation sequencing. More than 5000 transcripts, including protein coding and non-coding transcripts, were identified from each RIP-seq sample.
Project description:We report the result of lncRNAs binding to Hes1 in colon cancer SW480 cells using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, MD, USA) following the manufacturer's instructions, the RNAs were analyzed by Illumina HiSeq 2500 Sequencing.
Project description:In order to screen for CaMKIIβ binding lncRNAs, we performed a RIP-seq analysis on mouse hippocampus. Potential CaMKIIβ binding RNAs are identified through differential expression analysis between CaMKIIβ RIP-seq results and the control groups, IgG and input. Differentially expressed genes are subsequently filtered by biotype and expression level.
Project description:We performed RNA binding protein immunoprecipitation (RIP) assay with anti-IgG/anti-MATR3 antibody. The RNA libraries were constructed using rib-off and strand-specific kits. Next-generation sequencing (NGS) were performed to analyze MATR3-binding RNAs.
