CUTANA™ E. coli Spike-in DNA
63 in stock
Powered by BiozFragmented DNA derived from Escherichia coli (E. coli) can be used as a spike-in control for experimental normalization in Cleavage Under Targets and Release Using Nuclease (CUT&RUN). CUT&RUN normalization was originally performed with carryover E. coli DNA from MNase preparation [Meers et al., 2019], however pure preps of enzyme require post hoc DNA spike-in. CUTANA™ E. coli Spike-in DNA contains sufficient material for 100-200 CUT&RUN reactions (range for high and low abundance targets).
Figure 1: DNA gel data
E. coli fragment size distribution. Lane 1: gDNA extracted from JM101 E. coli cells (500 ng). Lane 2: Digested and purified CUTANA™ E. coli Spike-in DNA (500 ng) resolved via 2% E-Gel™ EX Agarose Gel. Migration positions of DNA molecular weight markers are indicated.
Figure 2: CUT&RUN sequencing data
CUTANA™ E. coli Spike-in DNA (1.0 ng) was added to CUT&RUN reactions using 500,000 K562 cells enriched for a low abundance target (H3K4me3, EpiCypher 13-0041), a high abundance target (H3K27me3, EpiCypher 13-0055) and an 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. NOTE: Spike-in DNA amount should be optimized by the end user with the goal of E. coli DNA comprising ~1% (0.2 – 5%) of total sequencing reads.
Application Notes
Prior to opening, pellet DNA by quickly spinning in a benchtop microfuge. Reconstitute in 200 µL DNase free water (0.5 ng/µL). Vortex tube on all sides to ensure complete resuspension. Quick spin in benchtop microfuge prior to use.
Use in CUT&RUN:
- Use with CUTANA™ pAG-MNase for ChIC/CUT&RUN (EpiCypher 15-1016), which has very low E. coli DNA carryover.
- Add 1-2 µL* (0.5-1 ng) E. coli Spike-in DNA directly to the Stop Buffer, which quenches calcium-mediated pAG-MNase DNA digestion. Gently vortex to mix, and add to samples.
*NOTE: Based on target abundance and antibody efficiency, the amount of E. coli DNA may need to be adjusted. Aim for spike-in DNA to comprise ~1% (0.2-5%) of total sequencing reads (Figure 2). - After sequencing, align reads to the reference genome of experimental samples (e.g. human) as well as to E. coli genome.
- Normalize data by following recommendations in the CUT&RUN Protocol.
