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When performing CUT&RUN with downstream NG-seq analysis, one can use a normal IgG antibody enriched sample as the negative control. However, we have seen and heard from other scientists that normal IgG antibodies generate very low diversity DNA libraries and can generate what looks to be specific enrichment of genomic regions in the CUT&RUN assay, complicating your NG-seq data analysis. Instead, we recommend generating and using an input chromatin DNA sample as described in the protocol of our CUT&RUN Assay Kit #86652. This input DNA sample generates a more diverse DNA library and works better as a negative control for analysis by NG-seq.
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Due to the expected and typically low yield of CUT&Tag DNA, we recommend using all 30 µL of the CUT&Tag DNA sample (from Section VI of the CUT&Tag protocol #77552) for library amplification. While CUT&Tag DNA libraries generated for histone modifications typically show some signal on the Agilent Bioanalyzer or TapeStation systems, libraries generated for non-histone proteins such as transcription factors and cofactors often have very weak or even no visible signal on Bioanalyzer or TapeStation systems, but they still generate NGS results with high-mapping rates, high numbers of identified binding peaks, and decent signal-to-noise ratios across the whole genome. For CUT&Tag libraries where the Bioanalyzer or TapeStation system is unable to identify the average size of the library, we suggest using a size o…
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Some CUT&RUN protocols utilize contaminating E. coli DNA in the pAG-MNase for sample normalization. However, this can become problematic when switching lots of pAG-MNase, because each lot of enzyme will have differing amounts of contaminating DNA. Instead, we recommend using a separate yeast spike-in DNA sample. We provide a yeast spike-in DNA sample with our CUT&RUN Assay Kit #86652 and our CUT&RUN pAG-MNase and Spike-In DNA #40366 for which every lot is tested and optimized for CUT&RUN. This yeast spike-in DNA provides better lot-to-lot consistency for your normalization efforts.