Enabling Epigenetics Research

CUT&Tag-IT® Spike-In Control, Anti-Mouse
Catalog No. 53173(Version A1)

Copyright 2024 Active Motif, Inc.

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Contents
Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2Workflow  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3CUT&Tag Spike-In Reaction Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4CUT&Tag Normalization Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8

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Overview
The Cleavage Under Targets and Tagmentation (CUT&Tag) technique has emerged as a
powerful method for investigating the genomic localization of histone modifications ofinterest. However, the identification of differences between data sets can be challenging
when global modification changes occur, such as in the case of studying the effects ofchromatin modifying enzyme inhibitors. Additionally inaccurate quantification of starting
material or technical variation during processing results in variation across sample data.
Currently available bioinformatic-based normalization methods are not applicable in these
instances, and the only reliable way to overcome bias and variation is to add a known
standard (Spike-In) into all samples. Active Motif offers Spike-In reagents for ChIP-Seq andhas now introduced a similar approach for CUT&Tag.
Active Motif’s strategy for CUT&Tag normalization is to Spike-In cryopreserved Drosophilacell nuclei into samples prior to CUT&Tag. Then, during the primary antibody incubation step,a Drosophila H2Av antibody is added in addition to the antibody targeting the histone markof interest. This Drosophila H2Av antibody provides a mechanism to reliably tag Drosophila
histones in a consistent way across all samples. A normalization factor is then created basedon the Drosophila signal and applied to the test genome. This CUT&Tag Spike-In strategy
enables normalization of CUT&Tag data independent of the experimental antibody andwithout bias.
The CUT&Tag-IT® Spike-In Control, Anti-Mouse works with mouse primary antibodies to thetarget of interest and the CUT&Tag-IT® Assay Kit, Anti-Mouse (Catalog No. 53165).
ProductFormat

Catalog No.

CUT&Tag-IT® Spike-In Control, Anti-Mouse

16 rxns

53173

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Kit Components and Storage
All components are guaranteed for 6 months after receipt when stored properly.
Kit Component

Quantity

Storage

CUT&Tag-IT® Spike-In Antibody, Mouse

16 µL

-20°C

CUT&Tag-IT Spike-In Nuclei

4 x 160 µL

-80°C

®

CUT&Tag-IT Spike-In Nuclei are provided as four 160 µL vials of nuclei at a concentration of500 nuclei/µL.
For abundant histone marks such as H3K27me3 we recommend a Spike-In:sample ratio of
1:10. For less abundant histone marks we recommend a Spike-In:sample ratio of 1:20.
Note:

If more than 1 vial of nuclei are used across an experiment, pool the neededvials of nuclei together to ensure consistency in the amount of nuclei added
per reaction for spike-in.

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Workflow

Cells

CUT&Tag-IT™ Spike-InNuclei

Target-Specific
Antibody

CUT&Tag-IT™ Spike-In
Antibody

Extract DNA

Amplify

Sequencing Library

Sequencing andAnalysis

Map to experimental
genome

Map to Drosophila
genomeNormalize Drosophila
tag counts across samples

Normalize experimental sample
tag counts by same ratio

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3

CUT&Tag Spike-In Reaction Guidelines
Active Motif’s CUT&Tag normalization strategy1 may be applied to any mammalian CUT&Tag
assay reaction due to the lack of cross-reactivity of the Spike-In antibody with mammaliansamples. The amount of cryopreserved Drosophila nuclei and antibody used per CUT&Tag
reaction may need to be optimized with the goal of having Drosophila reads make up only
2-10% of the total sequencing reads. However, when using robust antibodies against tightly
localized histone modifications, such as H3K4me3, we recommend a Spike-In:test sample
ratio of 1:20. For antibodies against spreading marks such as H3K27me3, we recommend at
Spike-In:test sample ratio of 1:10.
% DM / Total

Conditions

Human Cells

CUT&Tag-IT
Spike-In Nuclei

CUT&Tag-IT Spike-In
Nuclei/Human Cells Ratio

H3K4me3

H3K27me3

A

50K

20K

1:2.5

29.6%

16.9%

B

100K

20K

1:5

17.4%

8.9%

C150K

20K

1:7.5

12.6%

6.6%

D200K

20K

1:10

10.2%

5.0%

To demonstrate the utility of this approach, histone modification level differences weremimicked by setting up CUT&Tag reactions with different amounts of starting cell numbers(Figure 1). Various numbers of cryopreserved human K562 cells (50,000, 100,000, 150,000,and 200,000) were combined with 20,000 of cryopreserved Drosophila nuclei foreach experiment.
Two histone marks, H3K27me3 and H3K4me3, were evaluated in the CUT&Tag Spike-In
assay, with biological duplicates included in each experiment. Libraries were quantified andsequenced to a depth of 40-50 million reads per sample. However, sequencing to equal readdepth for each sample masks the differences in starting amounts for each sample (Figure 2).
Therefore Spike-In normalization is required to reveal the differences in starting material2. For
normalization, the sample with the lowest number of Drosophila reads (Figure 3) was used togenerate normalization factors across samples, which were then applied to down-sample thehuman read counts for each sample accordingly (Figure 4). After obtaining normalized humanread counts, a standard CUT&Tag pipeline was used for peak calling generation of bigWigs

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Fig. 1

Fig. 2

Fig. 3Fig. 4

Figure 1: K562 Starting Cell Numbers for CUT&Tag
50,000, 100,000, 150,000, and 200,000 K562 cells were used in CUT&Tag.
Figure 2: K562 Paired Alignments
Paired alignments from duplicate K562 samples were deduplicated with mitochondrial reads
removed. 50,000, 100,000, 150,000, and 200,000 K562 samples resulted in 3,879,516, 8,344,823,8,206,876, and 9,235,143 reads. The number of alignments is not reflective of the number of cells
added to the experiment, thus normalization is needed.
Figure 3: Drosophila Paired Alignments to Obtain Normalization Factor
20,000 CUT&Tag-IT Spike-In Nuclei were added to 50,000, 100,000, 150,000, and 200,000 K562
samples and assayed in CUT&Tag. The resulting Drosophila alignments for 50,000, 100,000,150,000, and 200,000 K562 samples were 1,620,351, 1,805,662, 1,206,376, and 1,054,504. Ingeneral, the more K562 cells in the experiment, the less Drosophila alignments resulted because
more reads were going to K562 cells. The sample with the lowest number of reads, in this case
200,000 K562 cells, was used for the normalization factor in Figure 4.
Figure 4: K562 Paired Alignments After Normalization to Drosophila Paired Alignments
K562 paired alignments were normalized to the Drosophila alignment number from 200,000
K562 cells shown in Figure 3. Now the expected ratios based on amount of starting cells has
been restored.

activemotif.com|5CUT&Tag Normalization Guidelines
1.

Perform CUT&Tag by combining the CUT&Tag-IT® Spike-In Nuclei with theexperimental sample cells to be used in CUT&Tag. Perform CUT&Tag as per theinstructions provide in Active Motif’s CUT&Tag-IT® Assay Kit, Anti-Mouse (Catalog No.
53165) and add 1 µL of CUT&Tag-IT® Spike-In Antibody, Mouse in combination withyour test antibody, during the primary antibody incubation step. Use the guidelines
provided for nuclei and antibody quantities based on the antibody target.

2.

Sequence the CUT&Tag libraries using an NGS platform.

3.

Map the CUT&Tag data to the test reference genome (e.g. human, mouse, or other).

4.

Map CUT&Tag data to the Drosophila reference genome.

5.

Count uniquely aligning Drosophila sequence tags and identify the sample containing
the least number of tags.

6.

Divide the aligned Drosophila tag value from the sample with the lowest Drosophila
tag count by the Drosophila tag count value from all other samples to and generatea normalization factor for each sample. (Sample 1 with lowest tag count / Sample 2)= Normalization factor. The sample with the lowest drosophila tag count will have anormalization factor of 1.

7.

Generate the normalization factors for all samples using the strategy from step 6.

8.

Use the normalization factors to down-sample the read counts for each sample.

9.

After obtaining normalized human read counts, use a standard CUT&Tag pipeline
starting with the downsampled tag counts for each sample for peak calling andgeneration of bigWigs.

activemotif.com|6References
1.

Egan, B. et al. (2016) PLoS ONE. 11(11): e0166438.

2.

Taruttis et al. (2017) Biotechniques 62:53-61

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