CUTANA™

CUTANA™ E. coli Spike-in DNA

$135.00
SKU: 18-1401
Pack size: 100 ng

Description

Fragmented 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 [1], 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 samples (range for high and low abundance targets).

Validation Data

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 samples using 500,000 K562 cells enriched for a low abundance target (H3K4me3, EpiCypher 13-0041), a high abundance target (H3K27me3, EpiCypher 13-0030) 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.

Technical Information

Formulation
100 ng lyophilized DNA. *NOTE: May not be visible.
Storage and Stability
Stable for 2 years at -20°C from date of receipt. After resuspending, aliquots should be stored at -20°C.

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:

  1. Use with CUTANA™ pAG-MNase for ChIC/CUT&RUN (EpiCypher 15-1016), which has very low E. coli DNA carryover.
  2. 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).
  3. After sequencing, align reads to the reference genome of experimental samples (e.g. human) as well as to E. coli genome.
  4. Normalize data by following recommendations in the CUT&RUN Protocol.

References

Background References:
[1] Meers et al. Elife. (2019). PMID: 31232687

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