Article Title: A New CUT RUN Low Volume-Urea (LoV-U) protocol uncovers Wnt/β-catenin tissue-specific genomic targets
Figure Lengend Snippet: CUT RUN with nuclear extraction and Urea-mediated release allows reproducible profiling of β-catenin binding in HEK293T. A. Genome coverage tracks of traditional CUT RUN (C R) for β-catenin and C R using urea in the elution buffer (C R-LoV-U: Low Volume-Urea, see Figure 2 for a complete explanation of the protocol) for β-catenin and IgG negative control, scaled to signal per million reads. Peak regions called by SEACR are shaded. C R-LoV-U shows enriched signal compared to traditional C R for β-catenin, and successfully recapitulates previously published ChIP-seq peaks at known WNT responsive elements, indicated by red lines corresponding to the exact positions of the β-catenin peaks called in Doumpas et al., 2019 . B. Genome coverage tracks of 3 biological replicates of the C R-LoV-U β-catenin and IgG negative control, showing reproducible signal enrichment across replicates. C. Left: Venn diagram of number of peaks called by SEACR for β-catenin replicates. The peaks called in 2 of 3 replicates were considered high-confidence and used for downstream analyses. Right: Pie chart showing genomic region annotations for β-catenin peaks. Bottom: Signal enrichment plot displaying fold-change over IgG control for each replicate over the high-confidence peak regions. D. Motif analysis results for high-confidence β-catenin peak regions, showing significant enrichment for TCF/LEF binding motifs. E. Gene Ontology analysis of the peak-associated genes, where the odds ratio (ratio of input list to reference list, x-axis) and the statistical significance (y-axis) for groups of “GO-biological processes” are represented, shows enrichment for several WNT pathway-related mechanisms.
Article Snippet: Cells were washed three times in Nuclear Extraction (NE) buffer (HEPES-KOH pH-8.2 [20 mM], KCl [10 mM], Spermidine [0.5 mM], IGEPAL [0.05%], Glycerol [20%], Roche Complete Protease Inhibitor EDTA-Free), resuspended in 40 μl NE per sample and bound to 20 μl Magnetic ConA Agarose beads equilibrated in binding buffer.
Techniques: Binding Assay, Negative Control, Chromatin Immunoprecipitation