CST BLOG: Lab Expectations

The official blog of Cell Signaling Technology (CST) where we discuss what to expect from your time at the bench, share tips, tricks, and information.

CUT&Tag DNA Library Yield: What to Do if Yours Is Too Low to Detect with an Agilent Bioanalyzer or TapeStation System

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Chromatin profiling with methods like ChIP-Seq, CUT&RUN, and now CUT&Tag have significantly contributed to our understanding of disease mechanisms and how modulating epigenetic changes can be used to develop novel therapeutics. After DNA libraries are generated using one of these three techniques, the libraries can be quantitated using platforms like the NanoDrop or Qubit system, QC’d with instruments like the Bioanalyzer or TapeStation system, and analyzed with next-generation sequencing (NGS). But what happens if the CUT&Tag DNA library concentration or the signal generated by the Bioanalyzer or TapeStation system is low? Let’s explore how you can still successfully sequence your DNA library.

All DNA Library Quantitation Methods Are Not Equal

Bioanalyzer or TapeStation instruments are often used to establish DNA and RNA sample quality control. Before analysis, the library concentration can be determined using the Thermo Fischer Scientific Nanodrop instrument, Qiagen QIAxpert system, Thermo Fisher Scientific Qubit Fluorometric Quantification system, or PicoGreen assay. However, the measured yield of the amplified CUT&Tag DNA library can vary based on the method used. CST scientists measured the same DNA library with each method to provide the following CUT&Tag DNA library yield guidelines. Just keep in mind that you can still get quality NGS data even if yields are low.

 

Method Target Expected Concentration Recommendations
NanoDrop or QIAxpert System Histone 10–20 ng/µL If your DNA library concentration is >3 ng/µL, we recommend moving forward with NGS analysis even if the library QC signal on BioAnalyzer or TapeStation system is weak or invisible. Refer to the CST Troubleshooting guide if the library concentration is <3 ng/µL.
Co-Factor or
Transcription Factor
5–12 ng/µL
Qubit Fluorometric Quantification System or PicoGreen Assay Histone 3–10 ng/µL Concentrations may be too low to run on the BioAnalyzer or TapeStation system. We recommend that you still analyze the library with NGS if your positive control, like Tri-Methyl-Histone H3 (Lys4), generates the expected library yield and/or Bioanalyzer or TapeStation peaks, or qPCR QC on the CUT&Tag DNA library generates a good signal-to-noise ratio.
Co-Factor or
Transcription Factor
<1 ng/µL

 

Refer to the CUT&Tag FAQ page to view supporting data and/or for guidance on pooling samples with different yields together.

DNA Libraries Quality Control

The Bioanalyzer or TapeStation system gives you information regarding the sample concentration, average fragment size range, and sample purity. When the target of interest, such as a transcription factor, is not abundant in a cell or when only a small number of cells are available, the data from the Bioanalyzer or TapeStation instrument can indicate low library yields. In these situations, it can be hard to know whether to move forward with NGS, and many times researchers using ChIP-seq or CUT&RUN DNA libraries will opt not to proceed. However, baseline thresholds are lower using CUT&Tag—which begs the question: can I still successfully sequence my CUT&Tag DNA library even if the signal from my Bioanalyzer or TapeStation system is low?

 

CUT&Tag Can Deliver Quality NGS Data Even if the Signal from the Bioanalyzer or TapeStation Is Low

When working with a low abundance target or a small number of cells, we suggest running a positive control like Tri-Methyl-Histone H3 (Lys4) along with your DNA library to evaluate protocol success.1 In cases where the positive control signal is high but the target library signal is low, the CUT&Tag DNA library can still be successfully sequenced to provide important protein-DNA interaction data (Figure 1). Thus, we recommend sequencing your CUT&Tag DNA library even if you see very weak or even no visible peaks in the profile from the Bioanalyzer or TapeStation system.

 

Robust TCF4/TCF7L2 Sequencing Data Obtained from Three DNA Libraries with Low Signal from a Bioanalyzer System

Robust TCF4 Sequencing Data Obtained From Three DNA Libraries with Low Signal From a Bioanalyzer System

23-ETC-03854 CUT&Tag Bioanalyzer_Blog Image2

Figure 1. CUT&Tag was performed with HCT 116 cells and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569, with several sources of loaded pAG-Tn5. The amount of each enzyme used was based on the manufacturer’s recommendation. DNA libraries were prepared using the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina #47415. The figure (upper image) shows the profiles obtained from a Bioanalyzer system for the three CUT&Tag DNA libraries.

The same DNA libraries analyzed with the Bioanalyzer system were sequenced and NGS tracks are shown (lower image). NGS data shows equivalent binding across chromosome 8 (upper), including MYC (lower), a known target gene of TCF4/TCF7L2.

qPCR: An Alternative QC Method for Your CUT&Tag DNA Library

If you still have concerns about sequencing your CUT&Tag DNA library with a low signal result from the Bioanalyzer or TapeStation system, another option is to perform qPCR on the DNA library against known positive and negative loci to assess the enrichment of chromatin fragments as a QC step before NGS. It is important to note that the qPCR should be performed on the CUT&Tag DNA library and not on the CUT&Tag DNA prior to library preparation. CUT&Tag DNA is not compatible with qPCR analysis prior to library amplification because both tagmented small DNA and large DNA are present in the sample as a result of the nuclear membrane being broken open at the end of the CUT&Tag assay. The tagmented DNA is selectively enriched during DNA library PCR amplification, which effectively dilutes out the genomic DNA and makes the CUT&Tag DNA library amenable to qPCR analysis. Generating a good signal-to-noise ratio with qPCR is a strong indicator that you will also be able to successfully sequence your CUT&Tag DNA library.

 

CUT&Tag DNA Library Generates qPCR Data with Much Higher Signal-to-Noise Ratios

CUT&Tag DNA library generates qPCR data with much higher signal-to-noise ratios

Figure 2. CUT&Tag was performed with HeLa cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, using CUT&Tag Assay Kit #77552. DNA libraries were prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The enriched DNA was quantified by real-time PCR using positive primer sets against the known binding sites of H3K4me3, SimpleChIP® Human RPL30 Exon 3 Primers #7014 and human GAPDH exon 1 primers Exon 1 Primers, and the negative primer set against a known negative site of H3K4me3, SimpleChIP Human MyoD1 Exon 1 Primers #4490. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one. 

 

Bottom line—don't let low signal from a Bioanalyzer or TapeStation system stop you from sequencing your CUT&Tag Library DNA. You will likely be missing out on high-quality results.

 

Reference

  1. Kaya-Okur HS, Wu SJ, Codomo CA, et al. CUT&Tag for efficient epigenomic profiling of small samples and single cellsNat Commun. 2019;10(1):1930. Published 2019 Apr 29. doi:10.1038/s41467-019-09982-5

CST, Cell Signaling Technology, and SimpleChIP are trademarks or registered trademarks of Cell Signaling Technology, Inc. All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

CUT&Tag provided under a license from Active Motif, Inc. under U.S. Patent No. 10,689,643 and 9,938,524, foreign equivalents, and child patents deriving therefrom. For purchaser’s internal research use only. May not be used for resale, services, or other commercial use.

U.S. Patent No. 11,733,248, foreign equivalents, and child patents deriving therefrom.

U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom. 23-ETC-03854

Andrea Tu, PhD
Andrea Tu, PhD
Andrea Tu, PhD, is a Scientific Marketing Manager at Kallidus Group who geeks out when learning about the latest scientific trends and developments. She received a PhD in Molecular and Cell Biology from the University of California, Berkeley where she studied Smad post-translational modifications in the TGF-β signaling pathway. Over the course of 20 years, Andrea has worked at UC San Diego, the Salk Institute, Stanford, Agilent Technologies, and Bio-Techne in technical roles spanning R&D, sales support, and marketing.

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