Analyzing CUT&RUN data
CUT&RUN analysis methods are similar to those used for ChIP-seq datasets, with a few key differences. Briefly:
- Align raw reads to a reference genome using Bowtie2 . The Integrative Genomics Viewer (IGV)  and/or deepTools2  can be used to visualize enrichment (e.g. bigWig files graphed over a genome browser).
- For peak calling, EpiCypher frequently uses MACS2  and SICER , programs for ChIP-seq that work well for CUT&RUN . SICER can be adjusted for analysis of sharp enrichment peaks (e.g. H3K4me3) vs. broad areas of enrichment (e.g. H3K27me3) . Other options include SEACR , a peak caller designed for CUT&RUN data, and the CUT&RUNTools 2.0 pipeline, which is designed for CUT&RUN and CUT&Tag data, including analysis of single cells . It is recommended to test several programs and select the one that faithfully represents the target of interest.
- To determine signal over background, EpiCypher uses bedTools to calculate fractions of reads in peaks (FRiP) and compare FRiP scores from experimental samples vs. controls . Other tools can be applied for differential analysis and heatmap generation (e.g. DESeq2 , deepTools2 ).
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