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CUT&RUN Frequently Asked Questions

Questions

Answers

Is there a maximum number of cells that can be used in a single CUT&RUN assay?

We have found 100,000 cells per assay to be sufficient for enriching target loci for multiple histone modifications, transcription factors, and transcription cofactors. However, one can increase the number of cells to 250,000 per assay without scaling up beads, antibodies, or buffers.

What is the fewest number of cells I can use in a reaction with your CUT&RUN assay kit?

We have shown that our CUT&RUN Assay Kit #86652 works with as few as 12,500 cells for some histone modifications, transcription factors, and cofactors (see image). However, we can not guarantee that the kit will work for all target proteins with this small number of cells.

Normalization of CUT&RUN assay signals

Normalization of CUT&RUN assay signals: Normalization of CUT&RUN assay signals using spike-in DNA for qPCR analysis. CUT&RUN assays were performed using the CUT&RUN Assay Kit #86652 with a decreasing number of HCT 116 cells and either Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 (upper panels) or Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb #13499 (lower panels). Enriched DNA was quantified by real-time PCR using the SimpleChIP® Universal qPCR Master Mix #88989 and SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human β-Actin Promoter Primers #13653, SimpleChIP® Human β-Actin 3' UTR Primers #13669, and SimpleChIP® Human MyoD1 Exon 1 Primers #4490. The amount of immunoprecipitated DNA in each sample is represented as the signal relative to the total amount of input chromatin (percent input for 100,000 cells). Non-normalized enrichments are depicted in the left panels. The sample normalization spike-in DNA was added into each reaction proportionally to the starting cell number. Based on the difference of qPCR signals from spike-in DNA in each sample, CUT&RUN signals were normalized to the sample containing 100,000 cells. Normalized enrichments are depicted in the right panels.

Do you observe any differences in assay performance among different cell lines?

We have performed CUT&RUN on dozens of cultured adherent and suspension cell lines, both human and mouse. So far we have not noticed any significant difference in performance between these cell lines.

Do you have any specific advice for working with adherent cell lines? How about suspension cell lines?

With adherent cell lines, one first needs to detach the cells from the dish. We recommend using trypsin to detach the cells. Alternatively, you can use accutase, but the resulting cell pellet doesn't pack as well during centrifugation, so you tend to lose cells during the wash steps. Suspension cells are much easier. Just count the cells using a hemacytometer or auto cell counter and then collect the number of cells you need. We don't observe a significant difference in assay performance with adherent versus suspension cells.

Is it possible to perform CUT&RUN on tissue samples like heart, brain, or developing mouse tissues?

We have not yet tested our CUT&RUN Assay Kit on tissue samples, so we do not know how well it will work. To perform CUT&RUN on tissue, one must first disaggregate the tissue into a single-cell suspension prior to performing CUT&RUN. In a publication by Derek H. Janssens et al. (2018; PMID30577869), the authors perform CUT&RUN on brain tissue that was snap frozen, thawed, and disaggregated in CUT&RUN wash buffer prior to performing the CUT&RUN assay. It is fairly easy and quick to disaggregate brain tissue into single cells. For other more fibrous tissues, we would recommend an appropriate fixation prior to disaggregation and performing CUT&RUN and a cross-link reversal step after isolating the digested DNA from the cells.

Does CUT&RUN work for plant cells? Can you share any insight you may have?

We have not tested our CUT&RUN Assay Kit using plant cells. However, Xiao-Yu Zheng et al. (2019; PMID30719569) describes a method for pre-treating plant tissues and preparing the nuclei for CUT&RUN. Our kit can be used downstream of this nuclei preparation protocol. In this case, digitonin may not be required for cell permeabilization during the CUT&RUN assay, but it may help further permeabilize the cell membrane if the plant cells are not 100% homogenous.

Do we need to optimize the MNase digestion conditions for different cell types?

Our optimized digestion conditions for the CUT&RUN assay work well across multiple cell lines, both adherent and suspension. In addition, we haven't observed significant changes in chromatin digestion when increasing or decreasing either the amount of pAG-MNase added or the time of digestion. Our observations are similar to those published by Peter Skene et al. (2018; PMID29651053).

Does your CUT&RUN Assay Kit work with isolated nuclei?

Our CUT&RUN Assay Kit is optimized for use with whole cells and works with a large number of cell lines, both adherent and suspension. While it is not typically necessary to pre-isolate cell nuclei for CUT&RUN, our kit should work with pre-isolated nuclei because the Concanavalin A beads bind to glycoproteins on the surface of both cells and nuclei. Pre-isolation of cell nuclei should only be necessary for cells that are not efficiently permeabilized by digitonin, such as yeast or plant cells that both contain cell walls.

Does CUT&RUN show a bias toward euchromatin or heterochromatin?

No. We don't observe any bias toward euchromatin or heterochromatin in our assays. In CUT&RUN, the target-specific antibody recruits the pAG-MNase and directs digestion to chromatin that is directly adjacent to the antibody, whether that be in euchromatin or heterochromatin. In this case, the active tethering of pAG-MNase to the chromatin allows for digestion to occur even in less accessible heterochromatin. Our CUT&RUN Assay Kit #86652 has been shown to work well with antibodies against active, accessible euchromatic histone modifications (see Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751) and activating transcription factors (see NF-kB p65 [D14E12] XP® Rabbit mAb #8242)), as well as repressive, inaccessible heterochromatic histone modifications (see Tri-Methyl-Histone H3 [Lys27] [C36B11] #9733) and polycomb repressor complex proteins that are associated with inactive heterochromatin (see EZH2 [D2C9] XP® Rabbit mAb #5246 and RING1B [D22F2] XP® Rabbit mAb #5694).

Accessible Chromatin, Target: H3K4me3

Accessible Chromatin, Target: H3K4me3: CUT&RUN was performed with HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 12 (upper), including GAPDH (lower), a known target gene of H3K4me3.

Accessible Chromatin, NF-κB p65

Accessible Chromatin, Target: NF-κB p65: CUT&RUN was performed with HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb #8242, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 1 (upper), including LAMC2 (lower), a known target gene of NF-κB p65

Inaccessible Heterochromatin, Target: Tri-Methyl-Histone H3

Inaccessible Heterochromatin, Target: Tri-Methyl-Histone H3: CUT&RUN was performed with HeLa cells and Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 20 (upper), including TCEA2 gene (lower).

Inaccessible Heterochromatin, Target: Ezh2

Inaccessible Heterochromatin, Target: Ezh2: CUT&RUN was performed with NCCIT cells and Ezh2 (D2C9) XP® Rabbit mAb #5246, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 7 (upper), including HoxA genes (lower), a known cluster of EZH2 target genes.

Inaccessible Heterochromatin, Target: RING1B

Inaccessible Heterochromatin, Target: RING1B: CUT&RUN was performed with NCCIT cells and either RING1B (D22F2) XP® Rabbit mAb #5694 or Bmi1 (D42B3) Rabbit mAb #5856, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. RING1B and Bmi1 are both PRC1 components. The figures show binding across HOXD (upper) and COMMD3 (lower) genes.

What controls can I use to show my CUT&RUN experiment is working?

Whether performing CUT&RUN with downstream qPCR or NG-seq analysis, we always recommend using a CUT&RUN-validated positive control antibody like our Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 and a negative control antibody like our Rabbit (DA1E) mAb IgG XP® Isotype Control Antibody #66362. These antibodies can be paired with our SimpleChIP® Human RPL30 Exon 3 Primers #7014 or SimpleChIP® Mouse RPL30 Intron 2 Primers #7015 to show that your CUT&RUN assay is working regardless of the performance of your desired target-specific antibody. All of these controls are included in our CUT&RUN Assay Kit #86652.

CUT&RUN qPCR Control Data

CUT&RUN qPCR Control Data: CUT&RUN and ChIP assays were performed with HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using this CUT&RUN Assay Kit #86652 (left panel) or the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 (right panel). The enriched DNA was quantified by real-time PCR using SimpleChIP® Human GAPDH Exon 1 Primers #5516 and SimpleChIP® Human α Satellite Repeat Primers #4486. 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.

CUT&RUN NGS Control Data

CUT&RUN NGS Control Data: CUT&RUN and ChIP assays were performed with HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, using this CUT&RUN Assay Kit #86652 or the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The upper panel compares enrichment of H3K4me3 across chromosome 12, while the lower panel compares enrichment at the GAPDH gene, a known target of H3K4me3. The input tracks are from the CUT&RUN input sample.

What is the best way to choose an antibody for my CUT&RUN assay if I can not find a CUT&RUN validated antibody for my target protein?

We recommend starting with a ChIP- or ChIP-seq-validated antibody; however, we have found that not all ChIP-validated antibodies work in the CUT&RUN assay. Alternatively, Peter J. Skene et al. (2017; PMID28079019) suggest that you may want to try an antibody validated for an immunofluorescence assay, as the antibody binding in CUT&RUN takes place in an intact nuclear environment, resembling conditions for antibody binding in immunoflourescence assays.

What type of spike-in DNA do you recommend?

Some protocols utilize contaminating E. coli DNA in the pAG-MNase for sample normalization. However, this can become problematic when switching lots of pAG-MNase, because each lot of enzyme will have differing amounts of contaminating DNA. Instead, we recommend using a separate yeast spike-in DNA sample. We provide a yeast spike-in DNA sample with our CUT&RUN Assay Kit #86652 and our CUT&RUN pAG-MNase and Spike-In DNA #40366 for which every lot is tested and optimized for CUT&RUN. This yeast spike-in DNA provides better lot-to-lot consistency for your normalization efforts.

How does the spike-in DNA allow for normalization of the CUT&RUN assay?

A specificied amount of pre-fragmented yeast genomic DNA is added to each CUT&RUN reaction after the digestion and prior to DNA purification. This spike-in component allows you to normalize for differences in DNA purification, qPCR, library preparation and sequencing efficiencies between samples. The protocol included in our CUT&RUN Assay Kit #86652 provides detailed instructions for the spike-in DNA control for both qPCR and NG-seq. It is important to note that significantly different amounts of spike-in DNA are required for qPCR vs. NG-seq, so please read the protocol carefully.

What is the expected size distribution of CUT&RUN DNA?

When using antibodies against histone modifications, we typically see fragment sizes as small as 150 bp (size of a mono-nucleosome) and quite often see a nucleosomal ladder of DNA fragment sizes (i.e. 150, 300, 450, 600 bp). When using antibodies against transcription factors, we see a range of DNA fragment sizes, with the majority being greater than 35 bp.

Target: H3K4me3

Target: H3K4me3: CUT&RUN assays were performed with HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The size of the DNA fragments in the library were analyzed using an Agilent Bioanalyzer®. The adaptor and barcode sequences added to the library during construction account for 140 bp in fragment length. As shown, excised DNA is highly enriched for mononucleosomes (peak around 300 bp).

Target: TCF4/TCF7L2

Target: TCF4/TCF7L2: DNA was purified using phenol/chloroform extraction followed by ethanol precipitation from a CUT&RUN assay performed using TCF4/TCF7L2 (C48H11) Rabbit mAb #2569. The size of the DNA fragments in the library was analyzed using a Bioanalyzer (Agilent Technologies). The adaptor and barcode sequences added to the library during construction account for 140 bp in fragment length.

I have read in the literature that when performing CUT&RUN for transcription factors I should purify my DNA using phenol-chloroform followed by ethanol precipitation, because small DNA fragments that are generated are lost during purification with DNA affinity columns. Is this correct?

We recommend using our DNA Purification Buffers and Spin Columns (for ChIP & CUT&RUN) #14209 for purifying your CUT&RUN DNA. These spin columns provide an easy and inexpensive method for purifying CUT&RUN DNA. In a side-by-side comparison, our DNA spin columns effectively recovered DNA fragments greater than, or equal to, 35 bp. While phenol-chloroform extraction followed by ethanol precipitation did more effectively recover smaller DNA fragments, when analyzing DNA libraries made from both samples, we found that the DNA columns captured approximately 98% of the total CUT&RUN DNA fragments. Approximately 2% of the DNA fragments found in the phenol-chloroform-extracted and ethanol-precipitated sample were less than 35 bp in length. Therefore, our DNA spin columns provide a simple, inexpensive, and robust method for purifying your CUT&RUN DNA fragments. Please note that not all DNA spin columns capture the same range of DNA fragment sizes, so if you use DNA spin columns from a different vendor, you will want to determine the minimum size of DNA fragment retained by those columns, as a higher fragment size cutoff can negatively impact your results.

Phenol/Chloroform Extraction vs. Spin Columns for DNA Purification

Phenol/Chloroform Extraction vs. Spin Columns for DNA Purification: Comparison of DNA purification using spin columns or phenol/chloroform extraction followed by ethanol precipitation. (A) A low range DNA ladder mix (lane 1, unpurified) was purified using either DNA Purification Buffers and Spin Columns (ChIP, CUT&RUN) #14209 (lane 2) or phenol/chloroform extraction followed by ethanol precipitation (lane 3) and separated by electrophoresis on a 4% agarose gel. As shown, phenol/chloroform followed by ethanol precipitation efficiently recovers all DNA fragment sizes, while DNA spin columns recover DNA fragments ≥35 bp. (B) DNA was purified using phenol/chloroform extraction followed by ethanol precipitation from a CUT&RUN assay performed using TCF4/TCF7L2 (C48H11) Rabbit mAb #2569. The size of the DNA fragments in the library was analyzed using a Bioanalyzer (Agilent Technologies). The adaptor and barcode sequences added to the library during construction account for 140 bp in fragment length. Therefore, starting 35 bp DNA fragments would be 175 bp in length after library preparation (indicated with blue vertical line in figure). As shown, less than 2% of the total CUT&RUN enriched DNA fragments are less than 175 bp (starting length of 35 bp), suggesting that DNA purification spin columns are sufficient for capture of >98% of the total CUT&RUN DNA fragments.

Can CUT&RUN enriched DNA be analyzed by qPCR, or do I have to do NGseq to analyze my data?

Our CUT&RUN Assay Kit #86652 is compatible with downstream qPCR analysis. The protocol with the kit provides instructions and reagents for generating an input DNA sample that can be used to quantify your CUT&RUN DNA by qPCR. For qPCR analysis of CUT&RUN DNA, we recommend designing primers that generate amplicons of 60 to 80 bp in length.

How do I QC my CUT&RUN DNA before spending money on NG-seq?

You can QC your CUT&RUN assay prior to NG-seq by performing qPCR analysis of your enriched DNA. Our CUT&RUN Assay Kit #86652 is compatible with downstream qPCR analysis.

What are the most suitable control DNA samples for my CUT&RUN-qPCR experiment?

When peforming CUT&RUN with downstream qPCR analysis, one can use a normal IgG antibody enriched sample as the negative control and calculate the target-specific antibody enrichment as fold over IgG background. However, the IgG sample typically contains extremely small amounts of DNA and is not a good indicator for how well your target-specific antibody is working in the assay. Therefore, we recommend generating and using an input chromatin DNA sample as well as described in the protocol of our CUT&RUN Assay Kit #86652. This way, you can express the target-specific antibody DNA enrichment as percent of the total input DNA and directly determine how well the target-specific antibody is working in the assay.

How much CUT&RUN DNA is required for NG-seq analysis?

This depends on the sensitivity of the DNA library prep kit. The typical yield for CUT&RUN DNA is 0.6 to 6 ng per reaction. Our SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795 will work with starting DNA as low as 0.5 ng. You can also increase the number of PCR cycles up to 20 to increase amplification of the library if the starting amount is too low.

What DNA library prep kit do you recommend using for CUT&RUN?

We recommend using our SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795 for making DNA libraries with CUT&RUN or ChIP DNA. This kit should be paired with our SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Dual Index Primers) #47538 or SimplechIP® ChIP-seq Multiplex Oligos for Illumina® (Single Index Primers) #29580.

Do you recommend performing size selection of the DNA during library preparation?

No, we don't recommend performing size selection during the library preparation for CUT&RUN or ChIP-seq. Based on our experience and feedback from customers, size selection can significantly derease both the yield and diversity of the DNA library. If you are concerned about large DNA fragements >1 kb, then simply decrease the extension time when amplifying the adaptor-ligated DNA sample during the libarary preparation. We find that reducing the extension time to 10 to 15 seconds greatly reduces amplification of large fragments > 1kb, while being sufficient for amplification of smaller and more desirable DNA fragments during the CUT&RUN library preparation.

What is the most suitable control DNA sample for my CUT&RUN NG-seq experiment?

When performing CUT&RUN with downstream NG-seq analysis, one can use a normal IgG antibody enriched sample as the negative control. However, we have seen and heard from other scientists that normal IgG antibodies generate very low diversity DNA libraries and can generate what looks to be specific enrichment of genomic regions in the CUT&RUN assay, complicating your NG-seq data analysis. Instead, we recommend generating and using an input chromatin DNA sample as described in the protocol of our CUT&RUN Assay Kit #86652. This input DNA sample generates a more diverse DNA library and works better as a negative control for analysis by NG-seq.

How did you determine that a NG-seq sequencing depth of 3 to 5 million sequencing reads was sufficient for CUT&RUN analysis?

This decision was based on our analysis of hundreds of CUT&RUN samples. We found that a sequencing depth lower than 3 million generated too few peaks and that the number of peaks increased with deeper sequencing. If desired, one can increase the sequencing depth to 10 million reads per sample. The duplication rate of sequencing reads significantly increases if the sequencing depth is >15 million reads per sample.

Sequencing Depth Comparison, Target H3K27me3

Sequencing Depth Comparison, Target H3K27me3: CUT&RUN was performed with HeLa cells and Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733, using CUT&RUN Assay Kit #86652. DNA libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. Same library DNA were sent for NGS at different sequencing depth as indicated. The figure shows binding across chromosome 11 (top tracks), MYOD1 gene (middle tracks) and the MYT1 gene (bottom tracks).

For any additional questions, please contact our Epigenetic Applications group directly at [email protected].

Christopher Fry, PhD
Director of Epigenetics Product Development

Fang Chen, PhD
Group Leader of Epigenetic Applications Team


posted January 2021

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