We recommend adding E. coli Spike-in DNA to all CUT&RUN reactions for sequencing normalization. The spike-in DNA is added following pAG-MNase digestion, as part of the Stop Buffer. The major goal is for E. coli DNA to comprise ~1% (0.2-5%) of total sequencing reads, which provides ample reads for normalization without overtaking the dataset.
The amount of E. coli DNA added per reaction should consider the number of cells per reaction, processing conditions (cross-linking vs. native), and target abundance. Thus, optimization may be required.
For reactions using 500,000 cells, add 0.5-1 ng E. coli Spike-in DNA. This amount of spike-in DNA reflects our standard CUTANA™ CUT&RUN protocol, and works for most cell types, targets, and processing conditions.
For lower cell numbers, the spike-in amount should be scaled linearly from the 500,000 cell number baseline. For instance, for 50,000 cells, use 0.05-0.1 ng E. coli Spike-in DNA.
Note that low-abundance targets often have higher a percentage of total sequencing reads assigned to E. coli DNA, while high-abundance targets may have a lower percentage of spike-in reads. As a result, a range of spike-in read coverage is acceptable (0.2-5%). This is nicely illustrated in Table 1 using our recommended CUTANA™ CUT&RUN control antibodies, which include low abundance (H3K4me3) and high abundance (H3K27me3) targets, as well as a negative control (IgG). Each reaction was performed using 500,000 K562 cells and spiked with 0.5 ng or 1.0 ng E. coli DNA. Observations are as follows:
IgG. The IgG negative control typically has a high percentage of reads attributed to E. coli DNA. In this experiment, 0.5 ng spike-in DNA was preferred (blue bold text), as it brings the percentage of E. coli reads below 5%.
H3K4me3. This low-abundance target needs only 0.5 ng spike-in DNA to generate the appropriate number of reads for normalization. However, 1.0 ng is also acceptable.
H3K27me3. As a high abundance target, H3K27me3 generates more unique sequencing reads, and thus has a lower percentage of reads assigned to E. coli DNA. In order to generate adequate reads for sequencing normalization, 1.0 ng of spike-in DNA is recommended (blue text).
E. coli Spike-in DNA | Target | Total reads | E. coli reads | % E. coli reads |
|---|---|---|---|---|
0.5 ng | IgG | 3,644,233 | 155,549 | 4.27% |
H3K4me3 | 3,121,112 | 42,210 | 1.35% | |
H3K27me3 | 5,254,299 | 8,511 | 0.16% | |
1.0 ng | IgG | 2,569,291 | 241,645 | 9.41% |
H3K4me3 | 3,127,912 | 147,565 | 4.72% | |
H3K27me3 | 9,650,258 | 22,419 | 0.23% |
Table 1: EpiCypher E. coli Spike-in DNA (0.5 and 1.0 ng) was added to CUT&RUN samples using 500,000 K562 cells enriched for a low abundance target (H3K4me3, EpiCypher 13-0060), a high abundance target (H3K27me3, EpiCypher 13-0055) and IgG negative control (EpiCypher 13-0042). Total numbers of paired-end sequencing reads, reads aligned to E. coli, and percentage of total sequencing reads aligned to E. coli spike-in DNA are shown. Blue bolded text highlights the spike-in amounts recommended for each target.