Chromatin profiling with strategies like ChIP-Seq, CUT&RUN, and now CUT&Tag have considerably contributed to our understanding of illness mechanisms and the way modulating epigenetic modifications can be utilized to develop novel therapeutics. After DNA libraries are generated utilizing certainly one of these three methods, the libraries might be quantitated utilizing platforms just like the NanoDrop or Qubit system, QC’d with devices just like the Bioanalyzer or TapeStation system, and analyzed with next-generation sequencing (NGS). However what occurs if the CUT&Tag DNA library focus or the sign generated by the Bioanalyzer or TapeStation system is low? Let’s discover how one can nonetheless efficiently sequence your DNA library.
All DNA Library Quantitation Strategies Are Not Equal
Bioanalyzer or TapeStation devices are sometimes used to ascertain DNA and RNA pattern high quality management. Earlier than evaluation, the library focus might be decided utilizing the Thermo Fischer Scientific Nanodrop instrument, Qiagen QIAxpert system, Thermo Fisher Scientific Qubit Fluorometric Quantification system, or PicoGreen assay. Nonetheless, the measured yield of the amplified CUT&Tag DNA library can fluctuate primarily based on the tactic used. CST scientists measured the identical DNA library with every technique to supply the next CUT&Tag DNA library yield tips. Simply needless to say you may nonetheless get high quality NGS information even when yields are low.
|NanoDrop or QIAxpert System
|In case your DNA library focus is >3 ng/µL, we advocate shifting ahead with NGS evaluation even when the library QC sign on BioAnalyzer or TapeStation system is weak or invisible. Confer with the CST Troubleshooting guide if the library focus is <3 ng/µL.
|Qubit Fluorometric Quantification System or PicoGreen Assay
|Concentrations could also be too low to run on the BioAnalyzer or TapeStation system. We advocate that you simply nonetheless analyze the library with NGS in case your constructive management, like Tri-Methyl-Histone H3 (Lys4), generates the anticipated library yield and/or Bioanalyzer or TapeStation peaks, or qPCR QC on the CUT&Tag DNA library generates a very good signal-to-noise ratio.
Confer with the CUT&Tag FAQ web page to view supporting information and/or for steering on pooling samples with totally different yields collectively.
DNA Libraries High quality Management
The Bioanalyzer or TapeStation system provides you data relating to the pattern focus, common fragment measurement vary, and pattern purity. When the goal of curiosity, equivalent to a transcription issue, isn’t considerable in a cell or when solely a small variety of cells can be found, the info from the Bioanalyzer or TapeStation instrument can point out low library yields. In these conditions, it may be exhausting to know whether or not to maneuver ahead with NGS, and lots of instances researchers utilizing ChIP-seq or CUT&RUN DNA libraries will choose to not proceed. Nonetheless, baseline thresholds are decrease utilizing CUT&Tag—which begs the query: can I nonetheless efficiently sequence my CUT&Tag DNA library even when the sign from my Bioanalyzer or TapeStation system is low?
CUT&Tag Can Ship High quality NGS Knowledge Even when the Sign from the Bioanalyzer or TapeStation Is Low
When working with a low abundance goal or a small variety of cells, we advise working a constructive management like Tri-Methyl-Histone H3 (Lys4) alongside together with your DNA library to judge protocol success.1 In circumstances the place the constructive management sign is excessive however the goal library sign is low, the CUT&Tag DNA library can nonetheless be efficiently sequenced to supply necessary protein-DNA interplay information (Determine 1). Thus, we advocate sequencing your CUT&Tag DNA library even for those who see very weak and even no seen peaks within the profile from the Bioanalyzer or TapeStation system.
Strong TCF4/TCF7L2 Sequencing Knowledge Obtained from Three DNA Libraries with Low Sign from a Bioanalyzer System
qPCR: An Different QC Technique for Your CUT&Tag DNA Library
CUT&Tag DNA Library Generates qPCR Knowledge with A lot Larger Sign-to-Noise Ratios
Determine 2. CUT&Tag was carried out with HeLa cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, utilizing CUT&Tag Assay Package #77552. DNA libraries had been ready utilizing CUT&Tag Twin Index Primers and PCR Grasp Combine for Illumina Programs #47415. The enriched DNA was quantified by real-time PCR utilizing constructive primer units in opposition to the identified binding websites of H3K4me3, SimpleChIP® Human RPL30 Exon 3 Primers #7014 and human GAPDH exon 1 primers Exon 1 Primers, and the unfavourable primer set in opposition to a identified unfavourable web site of H3K4me3, SimpleChIP Human MyoD1 Exon 1 Primers #4490. The quantity of immunoprecipitated DNA in every pattern is represented as sign relative to the entire quantity of enter chromatin, which is equal to 1.
Backside line—do not let low sign from a Bioanalyzer or TapeStation system cease you from sequencing your CUT&Tag Library DNA. You’ll seemingly be lacking out on high-quality outcomes.
Kaya-Okur HS, Wu SJ, Codomo CA, et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun. 2019;10(1):1930. Printed 2019 Apr 29. doi:10.1038/s41467-019-09982-5
CST, Cell Signaling Expertise, and SimpleChIP are emblems or registered emblems of Cell Signaling Expertise, Inc. All different emblems are the property of their respective homeowners. Go to cellsignal.com/trademarks for extra data.
CUT&Tag offered below a license from Energetic Motif, Inc. below U.S. Patent No. 10,689,643 and 9,938,524, international equivalents, and baby patents deriving therefrom. For purchaser’s inside analysis use solely. Is probably not used for resale, companies, or different business use.
U.S. Patent No. 11,733,248, international equivalents, and baby patents deriving therefrom.
U.S. Patent No. 7,429,487, international equivalents, and baby patents deriving therefrom. 23-ETC-03854