Figure 1. An improved fusion protein for CUT&RUN.

(A) Schematic diagram (not to scale) showing improvements to the pA-MNase fusion protein, which include addition of the C2 Protein G IgG binding domain, a 6-histidine tag for purification and a hemagglutinin tag (HA) for immunoprecipitation. (B) The Protein A/G hybrid fusion results in high-efficiency CUT&RUN for both rabbit and mouse primary antibodies. CUT&RUN for both rabbit and mouse RNAPII-Ser5phosphate using pAG/MNase were extracted from either the supernatant or the total cellular extract. Tracks are shown for the histone gene cluster at Chr6:26,000,000–26,300,000, where NPAT is a transcription factor that co-activates histone genes. Tracks for 2’ and 10’ time points are displayed at the same scale for each antibody and for both supernatant (supn) or total DNA extraction protocols.

Figure 1—figure supplement 1. An improved fusion protein for CUT&RUN.

(A) Plasmid map of pAG-ERH-MNase-6xHIS-HA. (B) Coomassie-stained gel of fusion protein eluted from nickel-agarose.

Figure 1—figure supplement 2. pAG/MNase titration.

(A) K562 cells were incubated with an antibody to H3K27me3 (CST #9733 Rabbit monoclonal), washed twice with 1 ml Dig-wash. The sample was split into aliquots for incubation with pA/MNase at the recommended concentration and a serial dilution of pAG/MNase, followed by 3 1 ml washes. After 30 min using the standard protocol, lImit digestions are seen at all dilutions for this abundant epitope, indicating that the amount of fusion protein used in this experiment was in excess. (B) Representative tracks from these samples on the same normalized count scale show consistently low CUT&RUN backgrounds with excess pAG/MNase, which indicates that washes are sufficient to minimize non-specific background cleavages. ENCODE ChIP-seq tracks are shown for comparison, where USC used CST #9733, and Broad Institute used Millipore 07–449.