A Universal Spike-In Normalization Strategy for CUT&RUN, CUT&Tag, and ATAC-Seq
Poster # 5328

Sarah Traynor*, Brad Townsley*, Shuxiong Wang, Soyoung Jeon, Julianna Webber, Brian Egan, and Benjamin Delatte
Active Motif, Inc., Carlsbad, CAIntroduction
The use of Spike-In controls as a means for normalization in NGS based assays has been
demonstrated to not only be useful but in some cases necessary to detect differences ingene expression or protein occupancy. The need and usefulness of Spike-Ins have been
demonstrated for RNA-Seq1, ChIP-Seq2, ATAC-Seq3, and other sequencing methodologies.
Multiple new methods have become commonplace in the field of chromatin biology including
CUT&Tag and CUT&RUN, however, normalization has not been demonstrated. Here we applied
Spike-In normalization to these techniques using Drosophila nuclei. Furthermore, we show thatfor these methods Spike-In is necessary when using treatments that confer global changes.

Spike-In Workflow
Spike-In Workflow
Cells

Spike-In Nuclei

Target-Specific
Antibody

Spike-In Antibody

Extract DNAAmplify

Sequencing Library

Sequencing andAnalysis

Map to experimental
genomeSpike-In nuclei are mixed with experimental cells prior toassay steps. After mixing, both the target and Spike-In
antibody are added to the sample. The assay is carried out
and both experimental and Spike-In DNA are isolated andprepared for sequencing. Reads mapping is performed to theexperimental and the Drosophila genome. For data analysis,
first a normalization factor (scale factor) is calculated basedon the number of reads aligned to the Spike-In genome. The
scale factor is then used to adjust the read depth, peak callingand bigWig files for each sample, compensating for sample-tosample variability.

Map to Drosophila
genomeNormalize Drosophila
tag counts across samples
Normalize experimental sample
tag counts by same ratio

Enabling Epigenetics Research

Global Changes Detected Using CUT&Tag withActive Motif Spike-In Technology

Global Changes Detected Using CUT&RUN with
Active Motif Spike-In TechnologyA)A)B)

B)

Comparison of CUT&Tag with and without Active Motif Spike-In Normalization Technology

Comparison of CUT&RUN with and without Active Motif Spike-In Normalization Technology

Global changes to H3K27me3 induced by GSK126 treatment are only detected when normalized withSpike-In nuclei, while H3K9me3, an unaffected epigenetic mark remains consistent in both data sets.
A) Genomic browser tracks generated using CUT&Tag data without Active Motif Spike-In normalization.
Purple tracks correspond to cells treated with DMSO, gold tracks cell treated with GSK126 for 3 days.
B) Genomic browser tracks generated using CUT&Tag data with Active Motif Spike-In normalization.
Purple tracks correspond to cells treated with DMSO, gold tracks cells treated with GSK126 for 3 days.

Global changes to p53 and EED induced by GSK126 treatment are only detected when normalized withSpike-In nuclei. A) Genomic browser tracks generated using CUT&RUN data without Active Motif SpikeIn normalization. Purple tracks correspond to cells treated with DMSO, gold tracks cell treated withGSK126 for 3 days. B) Genomic browser tracks generated using CUT&RUN data with Active Motif Spike-Innormalization. Purple tracks correspond to cells treated with DMSO, gold tracks cell treated with GSK126for 3 days.

Metagene Analysis for H3K27me3 and H3K9me3 (negative control) Enrichment

Metagene Analysis for p53 and EED Enrichment

A)

A)

B)

A)

B)

B)

A)

B)

Global reduction of H3K27me3 only detected
when using Spike-In normalization

No detectable change for H3K9me3 when usingSpike-In normalization

Global reduction of EED only detected whenusing Spike-In normalization

Global reduction of p53 only detected whenusing Spike-In normalization

Metagene analysis of CUT&Tag data for H3K27me3
generated A) without Spike-In normalization andB) with Spike-In normalization.

Metagene analysis of CUT&Tag data for H3K9me3
generated A) without Spike-In normalization andB) with Spike-In normalization.

Metagene analysis of CUT&RUN data for EED
generated A) without Spike-In normalization andB) with Spike-In normalization.

Metagene analysis of CUT&RUN data for p53
generated A) without Spike-In normalization andB) with Spike-In normalization.

Schematic of Experimental Treatment Causing Detectable Global ChangeA)

B)

GSK126 Causes Local but Not Global Changes in Chromatin Accessibility
A)

GSK126 Treatment
GSK126 inhibits Enhancer of zeste homolog 2 (EZH2), a methyltransferase subunit in the Polycomb
repressive complex 2 (PRC2) that tri-methylates H3K274. A) A schematic of a treatment with GSK126
showing lower levels of H3K27me3 detected in treated cells. B) A H3K27me3 western blot performed on
K562 cells that were treated with either GSK126 (+) or DMSO (-) for 3 days.

References
1. Risso, D., et al., 2014. Nat. Biotechnol. 31, 896-902.

B)

Global R-loop Changes Detected using CUT&Tag with
Active Motif Spike-in TechnologyA)

B)

Comparison of ATAC-Seq with and without Active Motif Spike-In Normalization Technology
Local changes in chromatin accessibility are observed at Polycomb protein gene targets but not athouse keeping gene loci suggesting GSK126 does not cause global changes. A) Genomic browser
tracks generated using ATAC-Seq data at HOXA gene cluster with and without Active Motif Spike-In
normalization. Purple tracks correspond to cells treated with DMSO, gold tracks cells treated with GSK126
for 3 days. B) Genomic browser tracks generated using ATAC-Seq data at GAPDH locus. Purple tracks
correspond to cells treated with DMSO, gold tracks cells treated with GSK126 for 3 days.

Metagene Analysis for ATAC-Seq EnrichmentA)

B)

Comparison of CUT&Tag data of RNase A digested permeabilized cells with and without Active MotifSpike-In Normalization Technology
Global changes to R-loops induced by RNase A digestion are only detected when normalized with Spike-Innuclei, while CTCF, an unaffected target, remains unchanged in both data sets. A) Genomic browser tracks
generated using CUT&Tag data without Active Motif Spike-In normalization. Purple tracks correspond toundigested cells, gold tracks cells digested with RNase A. B) Genomic browser tracks generated using
CUT&Tag data with Active Motif Spike-In normalization. Purple tracks correspond to undigested cells, gold
tracks cells digested with RNase A.

Metagene Analysis for R-loop and CTCF Enrichment

2. Egan, B., et al., 2016. PLoS ONE. 11, e0166438.

A)

B)

A)

B)

3. Stewart-Morgan, K., et al., 2019. Mol. Cell. 75, 284-297.
4. Zhao, Y., et al., 2019. EMBO. 38, e99599.

Summary
Active Motif’s Spike-In control acts as an internal reference that is not sensitive to treatment,
therefore the proportion of Spike-In reads in libraries can be used as a normalizing factor. Global
enrichment changes are only detectable in CUT&Tag and CUT&RUN when paired with Active Motif
Spike-In technology.

activemotif.com

No global increase of chromatin accessibility detected for GSK126 treated cells
Metagene analysis of ATAC-Seq data generated A) without Spike-In normalization and B) with Spike-In
normalization. Spike-In normalization gives confidence that changes are not global.

Global reduction of R-loops by RNase A digestion
only detected when using Spike-In normalization

No detectable change for CTCF after RNase A
digestion when using Spike-In normalization

Metagene analysis of R-loop CUT&Tag datagenerated A) without Spike-In normalization and B)
with Spike-In normalization.

Metagene analysis of CUT&Tag data for CTCF
generated A) without Spike-In normalization and B)
with Spike-In normalization.

Watch Active Motif webinars at activemotif.com/webinars

Contact: bdelatte@activemotif.com