Project description:To identify RNAs that bind to LC3B, we employed n-RIP-seq. After obtaining the nRIP-seq data and applying a rigorous threshold for peak calling, we detected 5,285 significant peaks associated with LC3B. The coverage analysis revealed a mean coverage of 99% for LC3B-associated peaks, suggesting widespread binding throughout the transcriptome
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: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:To identify the target mRNAs of the m6A reader proteins YTHDF1 and YTHDF2, we carried out anti-YTHDF1 and anti-YTHDF2 RNA Immunoprecipitation (RIP) followed by RNA-sequencing. Using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore), RNA from E12.5 wild-type mouse cortices and P0 wild-type mouse retinas was pulled down by rabbit polyclonal anti-YTHDF1 (proteintech) and rabbit polyclonal anti-YTHDF2 (proteintech), and then sequenced on Illumina HiSeq3000 platform. The filtered reads were mapped to the mouse reference genome (GRCm38) using STAR v2.5 with default parameters. The resulting bam files were fed to the HTSeq tool to count the number of RNA-seq reads, which was further normalized to calculate FPKM. To determine which gene is enriched, we computed the FPKM from RIP elute to input, and any fold change greater than 2 was considered enriched. From the embryonic cortex, we identified 986 and 1860 mRNAs by anti-YTHDF1 and anti-YTHDF2 RIP-seq, respectively. Anti-YTHDF1 and anti-YTHDF2 RIP-seq in mouse retina identified 2969 and 1638 mRNAs, respectively. This study provides the gene lists which show mRNAs binding with YTHDF1 and YTHDF2 in the mouse cortex and retina.