CUTANA™ E. coli Spike-in DNA

SKU: 18-1401
Pack size: 100 ng
Untitled Document
CUTANA E. coli Spike-in DNA 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). CUTANA E. coli Spike-in DNA contains sufficient material for 100-200 CUT&RUN samples (high and low abundance targets, respectively).

CUTANA E. coli Spike-in DNA Description Formulation: 100 ng lyophilized DNA. *NOTE: May not be visible.

CUTANA E. coli Spike-in DNA Description Storage and Stability: Stable for 2 years at -20°C from date of receipt. After resuspending, aliquots should be stored at -80°C.

CUTANA E. coli Spike-in DNA Description Application Notes:
Prior to opening, pellet DNA by quick spin in a benchtop microfuge. Reconstitute in 200 µL DNase free water (0.5 ng/µL). Vortex tube on all sides to ensure complete resuspension.

Use in CUT&RUN:
1. Use in combination with CUTANA pAG-MNase for ChIC/CUT&RUN (EpiCypher 15-1016), which has very low levels of E. coli DNA.
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 (see protocol: Gently vortex to mix, and add to CUT&RUN 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 (see Table, right).
3. After sequencing, align reads to the reference genome from the experimental samples (e.g. human) as well as to the E. coli genome.
4. Normalize data by following recommendations described in the EpiCypher CUTANA CUT&RUN Protocol:


View technical datasheet for this product. 18-1401 Datasheet

18-1401 Gel Data

DNA Gel Data: Lane 1: gDNA extracted from JM101 E. coli cells (500 ng) and 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.
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18-1401 Sequencing Data

CUT&RUN Sequencing Data: CUTANA 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 Catalog No. 13-0041), a high abundance target (H3K27me3, EpiCypher Catalog No. 13-0030) and IgG negative control (EpiCypher Catalog No. 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. Green boxes highlight the recommended E. coli DNA spike-in amounts for each target.
(Click image to enlarge)

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