CUT and RUN pAG-MNase and Spike-In DNA

The CUT&RUN pAG-MNase and Spike-In DNA kit provides enough enzyme and spike-in DNA to support 50 CUT&RUN assays. The pAG-MNase Enzyme #57813 is a fusion of Protein A and Protein G to Micrococcal Nuclease, and is recombinantly produced in E. coli. The pAG-MNase is compatible with multiple species of antibodies, including both rabbit and mouse. The Sample Normalization Spike-In DNA (10 pg/µl) #29987 is fragmented genomic DNA from the yeast S. cerevisiae that can be added to a CUT&RUN reaction to facilitate normalization between samples during NG-seq analysis.

Product Usage Information

For the pAG-MNase Enzyme, after cell permeabilization and primary antibody binding, resuspend cells in 50 μl of digitonin buffer containing 1.5 μl of pAG-MNase Enzyme (33X dilution). Incubate cell samples with rotation at 4°C for 1 hour, wash cells with digitonin buffer, and then perform the chromatin digestion. Sample Normalization Spike-In DNA can be added directly to the digestion stop buffer. For sample normalization with NG-seq, add 5 μl (50 pg) of Sample Normalization Spike-In DNA to each reaction. When using 100,000 cells or 1mg of tissue per reaction this ensures that the normalization reads are around 0.5% of the total sequencing reads. If more or less than 100,000 cells or 1mg of tissue are used per reaction, proportionally scale the volume of Sample Normalization Spike-In DNA up or down to adjust normalization reads to around 0.5% of total reads. When performing sample normalization, be sure to map the CUT&RUN sequencing data for all samples to both the test reference genome (e.g. human) and the sample normalization genome (S. cerevisiae).