Name: Genome-Wide Mapping of Histone Modifications and Transcription Factor Binding Sites in Neuroendocrine Small Cell Lung Cancer Cell Lines Using CUT and RUN (Video) | JoVE Journal
Uploaded: 2026-04-03T12:30:00.000+00:00
Duration: 11 min 50 s
Description: Discover CUT&RUN-seq for genome-wide mapping of histone modifications and transcription factor binding in small cell lung cancer cell lines. This low-input method reveals epigenetic regulatory elements and potential therapeutic targets.

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5 minutes

This research was supported by NCI K08CA241309 (PI: Hui-Zi Chen), ACS IRG Award (PI: Hui-Zi Chen), and American Lung Association Lung Cancer Discovery Award (PI: Hui-Zi Chen). Sequencing was performed at the Linda T. and John A. Mellowes Center for Genomic Sciences and Precision Medicine. We also thank all laboratory members for their insights and support of this work.

NOTE: This protocol (Figure 1) provides step-by-step instructions for performing CUT&#38;RUN-seq in SCLC NE cell lines, which typically grow as non-adherent aggregates or floating clusters.
1. Cell Culture
<ol>
	<li>Maintain cell lines DMS79 (ASCL1 subtype), H146 (ASCL1 subtype), H82 (NEUROD1 subtype), and H446 (NEUROD1 subtype) in 13 mL of RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (see Table of Materials) in 75 mL flasks. Cultivate the cells in an incubator at 37 &#176;C with 5% CO2. 
	NOTE: All cells used in this study were purchased from ATCC and have been authenticated with short tandem repeat profiling.</li>
</ol>
2. Cell permeabilization
NOTE: Proper cell permeabilization is essential for enabling antibodies, pAG-MNase, and digested protein-DNA fragments to freely diffuse in and out of the cells. Before starting the CUT&#38;RUN protocol on a new cell line, optimizations should be performed to determine the ideal digitonin concentration for effective permeabilization. The lowest concentration to achieve more than 95% permeabilization (95% of cells &#34;dead&#34;) should be used for the CUT&#38;RUN protocol.
<ol>
	<li>Place the 5% digitonin stock, protease inhibitor, and spermidine on ice and allow them to thaw completely.</li>
	<li>Prepare the Wash Buffer according to Table 1 and keep the buffer on ice .</li>
	<li>Prepare a 0.05% digitonin working solution by mixing 10 &#181;L of the 5% stock with 990 &#181;L of Wash Buffer. Mix thoroughly by vortexing and keep on ice.</li>
	<li>Generate a dilution series of digitonin (0.05%, 0.01%, 0.001%, and 0.0001%)&#8221;by sequentially diluting the previous solution to a final volume of 1 mL of each tube. Vortex each dilution before proceeding to the next. At the same time, prepare a 0.05% Dimethyl Sulfoxide (DMSO) control in Wash Buffer (100 &#181;L total; see Table 2). 
	NOTE: Digitonin and DMSO are hazardous chemicals. Handle with caution and discard all waste in a properly labeled hazardous-waste container with secure lids.</li>
	<li>Keep all digitonin dilution solutions and the DMSO control on ice until they are used.</li>
	<li>Determine the number of cells needed by multiplying the number of permeabilization conditions by the number of cells required per test. Include an additional 20% to compensate for pipetting variation (e.g., for five conditions at 5&#215;105 cells each, prepare approximately 1.2 x 5 x 5 x 105&#160;cells).</li>
	<li>Collect the required number of cultured cells by centrifugation at 600 &#215; g for 3 min at room temperature (RT). Resuspend the pellet in 600 &#181;L of 1&#215; PBS (100 &#181;L per condition plus 20% extra).</li>
	<li>Aliquot 100 &#181;L of the PBS-resuspended cells into five 1.5 mL microcentrifuge tubes- one tube for each digitonin concentration and one for the DMSO control.</li>
	<li>Centrifuge the tubes at 600 &#215; g for 3 min at RT.</li>
	<li>Carefully remove the supernatant and resuspend each pellet in 50 &#181;L of the corresponding digitonin or DMSO control buffer.</li>
	<li>Incubate the samples at RT for 10 min.</li>
	<li>Mix 10 &#181;L of the treated cells with 10 &#181;L of trypan blue and load 10 uL of the mixture onto a cell counting slide.</li>
	<li>Use a cell counter to quantify viable cells (unstained) and non-viable, permeabilized positive cells (stained blue).</li>
	<li>Identify the lowest digitonin concentration that produces the highest proportion of trypan blue positive cells. Use this concentration for the downstream CUT&#38;RUN experiment. Confirm that the DMSO control maintains viability typical of a normal healthy cell culture.</li>
</ol>
3. CUT&#38;RUN seq
NOTE: The reagents in this protocol are scaled for 5 reactions, with extra added to account for pipetting loss. For a list of materials and equipment, see Table of Materials. At least two independent biological replicates are recommended for each CUT&#38;RUN experiment.
<ol>
	<li>Day 1
	<ol>
		<li>CUT&#38;RUN buffer prep
		<ol>
			<li>Prepare the Wash buffer by combining Pre-Wash Buffer, 25x Protease Inhibitor, and 1 M Spermidine as outlined in Table 3.</li>
			<li>Prepare the Cell Permeabilization Buffer by adding 5% Digitonin to the Wash Buffer according to Table 3.</li>
			<li>Prepare the Antibody Buffer by adding 0.4 &#181;L of EDTA to 100 &#181;L of Cell Permeabilization Buffer with the appropriate digitonin concentration, as described in Table 3. Place the prepared buffer on ice.</li>
			<li>Store the remaining Cell Permeabilization Buffer at 4 &#176;C for use on Day 2.</li>
		</ol>
		</li>
		<li>Concanavalin A (ConA) beads activation
		<ol>
			<li>Gently resuspend ConA beads suspension to ensure its even distribution.</li>
			<li>Transfer 55 &#181;L of the beads suspension (11 &#181;L per sample) into a 1.5 mL microcentrifuge tube.</li>
			<li>Place tube on a 1.5 mL magnetic rack until the beads have fully separated, then pipette to remove supernatant.</li>
			<li>Immediately wash the beads twice with 500 &#181;L of ice-cold Bead Activation Buffer (100 &#181;L per sample). Resuspend the beads by gentle pipetting for each wash and use the magnetic rack to discard supernatant between washes.</li>
			<li>After the final wash, remove the supernatant and gently resuspend the activated beads in 55 &#181;L (11 &#181;L per sample) of ice-cold Bead Activation Buffer.</li>
			<li>Aliquot 10 &#181;L of the beads suspension into each tube of an 8-strip tube set and keep the tubes on ice.</li>
		</ol>
		</li>
		<li>Harvest cells and bind cells to activated beads 
		NOTE: Cell viability is important for the signal/background ratio. However, the viability of SCLC NE cell lines can be variable (70%-90%) due to inherently high rate of cell turnover and preference to grow as medium-large aggregates in suspension.
		<ol>
			<li>Remove the cells from the 37 &#176;C incubator and examine them under a microscope to ensure quality.</li>
			<li>Transfer the cell suspension to a 50 mL conical tube and centrifuge at 1,000-1,200 &#215; g for 5 min at RT.</li>
			<li>Aspirate the medium from the conical tube and rinse the cell pellet with 20 mL of 1&#215; PBS.</li>
			<li>Centrifuge again, aspirate the PBS, and digest the pellet with Accutase for 3-5 min at 37&#176;C, based on the size of the pellet.</li>
			<li>Terminate the digestion by adding 5 mL of PBS + 10% FBS. Obtain the single-cell suspension by gently pipetting up and down with a serological pipette.</li>
			<li>Centrifuge the cells at 1,000-1,200 &#215; g for 5 min, discard the supernatant, and resuspend the cell pellet in 5 mL of PBS.</li>
			<li>Count starting cells, confirm cell viability and integrity.</li>
			<li>Calculate the total number of cells needed by multiplying the number of reactions by the number of cells per reaction (5&#215;105). Add 20% excess to account for pipetting errors (e.g., 1.2 &#215; 6 &#215; 5 &#215; 105 for 6 reactions). Transfer the calculated cell suspensions to 1.5 mL tubes. Centrifuge at 600 x g, 3 min at RT. 
			NOTE: Prepare at least one additional reaction as a backup.</li>
			<li>Resuspend the cells in RT Wash Buffer at a density of 5&#215;105 cells per reaction (six reactions total), using 100 &#181;L per reaction, gently but thorough pipetting to ensure a uniform suspension. Centrifuge at 600 x g for 3 min at RT. Pipette to remove supernatant.</li>
			<li>Repeat the wash step (Step 9) once more.</li>
			<li>Aliquot 100 &#181;L of washed cells to each tube in the 8-strip set containing 10 &#181;L of activated CoA beads. Gently pipette to mix and quickly spin in a mini centrifuge to collect the slurry (beads should not settle).</li>
			<li>Incubate bead-cell slurry for 10 min at RT to allow cells to bind to the beads.</li>
			<li>Place tubes on a 0.2 mL tubes magnet rack, allow the slurry to clear, and pipette to remove supernatant. Save 10 &#181;L of supernatant to confirm cells are bound to beads. Discard the remaining supernatant and move quickly to the next step.</li>
			<li>Remove tubes from the magnet. Immediately add 50 &#181;L of ice-cold Antibody Buffer to each reaction and gently pipette to resuspend the beads.</li>
			<li>Mix 10 &#181;L of both the supernatant and 10 &#181;L of the cell/bead suspension (resuspended in Antibody Buffer) with 10 &#181;L of trypan blue and load the mixture onto the cell counter slide.</li>
			<li>Analyze with a cell counter. Confirm the supernatant contains almost no beads or cells, and the bead/cell mixture contains permeabilized cells surrounded by beads. 
			NOTE: It is important to check the Cells CoA beads binding compatibility.</li>
		</ol>
		</li>
		<li>Permeabilization and antibody binding 
		NOTE: The success of CUT&#38;RUN critically depends on the antibody&#39;s affinity for its target and its specificity under specific experimental conditions. We recommend the following: 1. Use commercial antibodies that have been validated for CUT&#38;RUN when available. 2. For targets without CUT&#38;RUN-validated antibodies, consider those validated for Immunofluorescence (IF), as IF antibodies recognize epitopes in their native cellular context, similar to in situ binding of antibody to target within permeabilized cells in CUT&#38;RUN. 3. Test 3-5 antibodies in parallel to identify the most effective one for your specific application.
		<ol>
			<li>Quick spin the K-MetStat Panel and pipette to resuspend. Add 2 &#181;L of K-MetStat Panel to reactions designated for H3K4me3 positive &#38; IgG negative control antibodies. Pipette to mix and quick spin tubes (Optional).</li>
			<li>Add 0.5 &#181;g each of H3K4me3, H3K4me1, and IgG antibodies, and 0.6 &#181;g of E2F7 polyclonal antibody from different sources to each reaction (Table 4). Label the 8-strip tubes.</li>
			<li>Gently pipette to properly mix each reaction tube. Incubate overnight at 4 &#176;C on a nutator with the tube caps elevated. Do not rotate or invert the tubes.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Binding of pAG-MNase to antibody 
		NOTE: Beads settling overnight is normal and does not affect the results. If the beads become clumpy or sticky after overnight incubation, resuspend the beads by gentle pipetting. To improve consistency and throughput, multi-channel pipetting is recommended. When using 8-strip tubes, remove and replace buffers one strip at a time to prevent ConA beads from drying out.
		<ol>
			<li>Put the magnet stand on ice.</li>
			<li>Remove the 8-strip tubes from 4 &#730;C incubation and quick spin to collect liquid.</li>
			<li>Place tubes on the magnet stand until the slurry clears. Carefully pipette and remove the supernatant.</li>
			<li>Keep tubes on the magnet stand. Wash tubes twice with 200 &#181;L of cold Cell Permeabilization Buffer. Pipette to remove supernatant.</li>
			<li>Remove tubes from the magnet stand and immediately add 50 &#181;L of cold Cell Permeabilization Buffer to each reaction. Gently pipetting to resuspend cells/beads.</li>
			<li>Add 2.5 &#181;L of pAG-MNase to each reaction and mix well by gently pipetting.</li>
			<li>Incubate reactions for 10 min at RT.</li>
			<li>Quick spin the tubes, place them back on the magnet stand, until the slurry clears. Pipette to remove supernatant.</li>
			<li>Keeping tubes on magnet stand, wash tubes twice with 200 &#181;L of cold Cell Permeabilization Buffer. Pipette to remove supernatant.</li>
			<li>Remove tubes from the magnet stand, gently resuspend in 50 &#181;L of cold Cell Permeabilization Buffer. Place the tubes on ice.</li>
		</ol>
		</li>
		<li>Targeted chromatin digestion and release 
		NOTE: Perform Ca2+-dependent digestion at 0 &#176;C to minimize unintended MNase cleavage of accessible DNA12. Include spike-in DNA, such as Escherichia coli (E. coli), so that it represents approximately 0.5-5% (ideally ~1%) of total sequencing reads13. Spike-in DNA is used to normalize sequencing depth and is particularly important when comparing samples across a series, for example, when evaluating the global histone modification changes after inhibition or genetic deletion of histone modifying enzymes.
		<ol>
			<li>Add 1 &#181;L of 100 mM Calcium Chloride to each reaction in the 8-tube strip (from step 3.2.1.10) while on ice. Gently pipette to fully resuspend beads and ensure efficient digestion.</li>
			<li>Incubate the tubes on a nutator at 4 &#176;C for 2 h, with the capped ends elevated.</li>
			<li>In a new 1.5 mL tube, prepare the Stop Buffer Mix. For each reaction, combine 33 &#181;L of Stop Buffer with 1 &#181;L of E. coli spike-in DNA (0.5 ng). Gently vortex to mix.</li>
			<li>Add 33 &#181;L of stop buffer to each reaction at the end of the 2 h incubation. Mix by gentle pipetting.</li>
			<li>Incubate the reactions in a thermocycler at 37 &#176;C for 10 min.</li>
			<li>Quick spin tubes to collect the contents at the bottom, then place them on a magnet rack until the solution becomes clear.</li>
			<li>Carefully transfer approximately 84&#160;&#181;L supernatant which contains the CUT&#38;RUN-released DNA into fresh 1.5 mL tubes.</li>
		</ol>
		</li>
		<li>DNA purification using the DNA Purification Kit (see Table of Materials) 
		NOTE: Before first use, add 6.9 mL isopropanol to the DNA Binding Buffer and add 20 mL &#8805; 95% ethanol to the DNA Wash Buffer.
		<ol>
			<li>Add 420 &#181;L of DNA Binding Buffer to each collected supernatant. Vortex briefly to mix thoroughly.</li>
			<li>Place a DNA Cleanup column into its collection tubes.</li>
			<li>Load each sample onto the corresponding column.</li>
			<li>Centrifuge at 16,000 &#215; g for 30 s at RT. Discard the flow-through and return the column to the collection tube.</li>
			<li>Add 200 &#181;L of DNA wash buffer to each column and centrifuge at 16,000 x g for 30 s at RT. Discard the flow-through and replace the column in the collection tube.</li>
			<li>Repeat the washing step once.</li>
			<li>Centrifuge again at 16,000 x g for 30 s at RT to fully remove residual wash buffer.</li>
			<li>Transfer columns to labeled 1.5 mL tubes.</li>
			<li>Apply 12 &#181;L of Elution Buffer directly to the membrane at the center of the column.</li>
			<li>Incubate for 5 min at RT and then centrifuge at 16,000 &#215; g for 1 min at RT to elute the DNA. 
			NOTE: This is a safe stopping point. Store eluted DNA at -20 &#730;C for future processing.</li>
		</ol>
		</li>
		<li>CUT&#38;RUN DNA quantification and fragment size distribution analysis 
		NOTE: Quantify DNA using a Fluorometer with the dsDNA High Sensitivity assay Kit, which provides more accurate measurements of double-stranded DNA than absorbance-based methods.
		<ol>
			<li>Use 1 &#181;L of each sample to determine CUT&#38;RUN DNA concentration using the dsDNA High Sensitivity Assay Kit (see Table of Materials).</li>
			<li>Use an additional 1 &#181;L of DNA to assess the presence and size distribution of cleaved fragments. Perform fragment analysis using the High Sensitivity DNA Kit (see Table of Materials) following the manufacturer&#39;s guidelines (optional). 
			NOTE: Highly abundant binding proteins, such as histone post-translational modifications (PTMs), can be readily detected at this step. However, low-abundance transcription factors may be more difficult to detect.</li>
		</ol>
		</li>
		<li>Library construction, fragment analysis, and sequencing 
		NOTE: &#8804;5 ng total CUT&#38;RUN DNA, as determined by dsDNA High Sensitivity Assay Kit quantification, was used for CUT&#38;RUN next-generation sequencing (NGS) libraries&#39; construction. To minimize adapter dimers, reduce the Adapter from Illumina to 1 &#181;L for the adapter ligation. The number of PCR cycles during library construction can be adjusted based on input amount, library yield, and duplication rate. Different DNA purification Magnetic Beads (see Table of Materials) size selection strategies may be used depending on the target-histone PTMs or transcription factors.
		<ol>
			<li>Library construction was built by using the CUT&#38;RUN library prep kit (see Table of Materials) according to the kit manufacturer&#39;s instructions, with small modifications. In brief, 5 ng of CUT&#38;RUN DNA was used as the recommended input for library preparation. For DNA less than 5 ng, use as much as possible for library prep. Dual i5 &#38; i7 index primers were used for each sample to ensure successful multiplexed sequencing. 
			NOTE: This is a safe stopping point. The DNA can be stored at -20&#730;C for further processing.</li>
			<li>Measure the concentration of CUT&#38;RUN library with a Fluorometer and the dsDNA High Sensitivity Assay Kit. The size distribution of the CUT&#38;RUN NGS libraries was determined using the High Sensitivity DNA Kit (see Table of Materials) based on the High Sensitivity DNA Kit Guide or a comparable capillary electrophoresis instrument TapeStation (see Table of Materials). 
			NOTE: Dilute the CUT&#38;RUN library to bring the concentration within the detection range of the dsDNA High Sensitivity Assay Kit and the High Sensitivity DNA Kit.</li>
			<li>Pool libraries (40-46) based on equal molecular and unique indexes. After DNA purification Magnetic Beads size selection, Paired-end sequencing (100 bp read length) for the pooled libraries was performed on an Illumina NovaSeq 6000 system for half S-Prime (SP) at a read depth of estimated 10 million reads per library.</li>
		</ol>
		</li>
		<li>CUT&#38;RUN data analysis
		<ol>
			<li>Analyze CUT&#38;RUN data using a custom pipeline (Figure 5A). Assess read quality with FastQC. Remove adapter sequences from the start of the reads using Cutadapt. Align paired-end reads to the human genome (hg38) and the E. coli reference genome with Bowtie2. Use Samtools to remove unmapped reads. Remove duplicate reads with Picard, and generate bedGraph files using Bedtools. Create bigWig (bw) files with DeepTools. Visualize the data using IGV and DeepTools. Normalize total read counts to the spike-in E. coli read counts. Perform peak calling using SEACR (Sparse Enrichment Analysis for CUT&#38;RUN)19.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Kimura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+modifications+for+human+epigenome+analysis%5BTitle%5D)+AND+%22J+Hum+Genet%22%5BJournal%5D)">Histone modifications for human epigenome analysis.</a> J Hum Genet. 58 (7), 439-445 (2013).</li>
<li>Millan-Zambrano, G., Burton, A., Bannister, A. J., Schneider, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+post-translational+modifications-cause+and+consequence+of+genome+function%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Histone post-translational modifications-cause and consequence of genome function.</a> Nat Rev Genet. 23 (9), 563-580 (2022).</li>
<li>Seligson, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+histone+modification+patterns+predict+risk+of+prostate+cancer+recurrence%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Global histone modification patterns predict risk of prostate cancer recurrence.</a> Nature. 435 (7046), 1262-1266 (2005).</li>
<li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((STAT3+facilitates+super+enhancer+formation+to+promote+fibroblast-to-myofibroblast+differentiation+by+the+analysis+of+ATAC-seq%2C+RNA-seq+and+ChIP-seq%5BTitle%5D)+AND+%22J+Cell+Mol+Med%22%5BJournal%5D)">STAT3 facilitates super enhancer formation to promote fibroblast-to-myofibroblast differentiation by the analysis of ATAC-seq, RNA-seq and ChIP-seq.</a> J Cell Mol Med. 29 (11), e70639(2025).</li>
<li>Zhao, Z., Shilatifard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetic+modifications+of+histones+in+cancer%5BTitle%5D)+AND+%22Genome+Biol%22%5BJournal%5D)">Epigenetic modifications of histones in cancer.</a> Genome Biol. 20 (1), 245(2019).</li>
<li>Takumida, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrative+epigenome+and+transcriptome+analyses+reveal+transcriptional+programs+differentially+regulated+by+ASCL1+and+NEUROD1+in+small+cell+lung+cancer%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Integrative epigenome and transcriptome analyses reveal transcriptional programs differentially regulated by ASCL1 and NEUROD1 in small cell lung cancer.</a> Oncogene. 44 (34), 3113-3125 (2025).</li>
<li>Sivakumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrative+analysis+of+a+large+real-world+cohort+of+small+cell+lung+cancer+identifies+distinct+genetic+subtypes+and+insights+into+histologic+transformation%5BTitle%5D)+AND+%22Cancer+Discov%22%5BJournal%5D)">Integrative analysis of a large real-world cohort of small cell lung cancer identifies distinct genetic subtypes and insights into histologic transformation.</a> Cancer Discov. 13 (7), 1572-1591 (2023).</li>
<li>Rudin, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Author+correction%3A+Molecular+subtypes+of+small+cell+lung+cancer%3A+A+synthesis+of+human+and+mouse+model+data%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">Author correction: Molecular subtypes of small cell lung cancer: A synthesis of human and mouse model data.</a> Nat Rev Cancer. 19 (7), 415(2019).</li>
<li>Rhee, H. S., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+genome-wide+protein-DNA+interactions+detected+at+single-nucleotide+resolution%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution.</a> Cell. 147 (6), 1408-1419 (2011).</li>
<li>Teytelman, L., Thurtle, D. M., Rine, J., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+expressed+loci+are+vulnerable+to+misleading+ChIP+localization+of+multiple+unrelated+proteins%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins.</a> Proc Natl Acad Sci U S A. 110 (46), 18602-18607 (2013).</li>
<li>Skene, P. J., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+efficient+targeted+nuclease+strategy+for+high-resolution+mapping+of+DNA+binding+sites%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites.</a> Elife. 6, e21856(2017).</li>
<li>Skene, P. J., Henikoff, J. G., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+in+situ+genome-wide+profiling+with+high+efficiency+for+low+cell+numbers%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Targeted in situ genome-wide profiling with high efficiency for low cell numbers.</a> Nat Protoc. 13 (5), 1006-1019 (2018).</li>
<li>Meers, M. P., Bryson, T. D., Henikoff, J. G., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+CUT%26RUN+chromatin+profiling+tools%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Improved CUT&RUN chromatin profiling tools.</a> Elife. 8, e46314(2019).</li>
<li>Custers, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cleavage+under+targets+%26+release+using+nuclease+%28CUT%26RUN%29+protocols+for+the+study+of+genetic+and+epigenetic+regulation+in+cancer%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Cleavage under targets & release using nuclease (CUT&RUN) protocols for the study of genetic and epigenetic regulation in cancer.</a> Methods Mol Biol. 2944, 207-225 (2025).</li>
<li>Hainer, S. J., Boskovic, A., McCannell, K. N., Rando, O. J., Fazzio, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Profiling+of+pluripotency+factors+in+single+cells+and+early+embryos%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Profiling of pluripotency factors in single cells and early embryos.</a> Cell. 177 (5), 1319-1329.e11 (2019).</li>
<li>Marroquin-Rivera, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune-related+transcriptomic+and+epigenetic+reconfiguration+in+BV2+cells+after+lipopolysaccharide+exposure%3A+An+in+vitro+omics+integrative+study%5BTitle%5D)+AND+%22Inflamm+Res%22%5BJournal%5D)">Immune-related transcriptomic and epigenetic reconfiguration in BV2 cells after lipopolysaccharide exposure: An in vitro omics integrative study.</a> Inflamm Res. 73 (2), 211-225 (2024).</li>
<li>Huang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deciphering+the+role+of+histone+modifications+in+memory+and+exhausted+CD8+T+cells%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Deciphering the role of histone modifications in memory and exhausted CD8 T cells.</a> Sci Rep. 15 (1), 17359(2025).</li>
<li>De Sarkar, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nucleosome+patterns+in+circulating+tumor+DNA+reveal+transcriptional+regulation+of+advanced+prostate+cancer+phenotypes%5BTitle%5D)+AND+%22Cancer+Discov%22%5BJournal%5D)">Nucleosome patterns in circulating tumor DNA reveal transcriptional regulation of advanced prostate cancer phenotypes.</a> Cancer Discov. 13 (3), 632-653 (2023).</li>
<li>Roberti, A., Fraga, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenomic+profiling+of+histone+posttranslational+modifications+in+frozen+cancer+tissue+using+CUT%26RUN%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Epigenomic profiling of histone posttranslational modifications in frozen cancer tissue using CUT&RUN.</a> Methods Mol Biol. 2930, 229-243 (2025).</li>
<li>Black, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Master+regulators+and+cofactors+of+human+neuronal+cell+fate+specification+identified+by+CRISPR+gene+activation+screens%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Master regulators and cofactors of human neuronal cell fate specification identified by CRISPR gene activation screens.</a> Cell Rep. 33 (9), 108460(2020).</li>
<li>Augert, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+cell+lung+cancer+exhibits+frequent+inactivating+mutations+in+the+histone+methyltransferase+KMT2D%2FMLL2%3A+CALGB+151111+%28Alliance%29%5BTitle%5D)+AND+%22J+Thorac+Oncol%22%5BJournal%5D)">Small cell lung cancer exhibits frequent inactivating mutations in the histone methyltransferase KMT2D/MLL2: CALGB 151111 (Alliance).</a> J Thorac Oncol. 12 (4), 704-713 (2017).</li>
<li>Kaya-Okur, H. S., Janssens, D. H., Henikoff, J. G., Ahmad, K., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+low-cost+chromatin+profiling+with+CUT%26Tag%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Efficient low-cost chromatin profiling with CUT&Tag.</a> Nat Protoc. 15 (10), 3264-3283 (2020).</li>
<li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((H3K9me3+involved+in+FOXL2+regulation+of+SerpinE2+expression+to+affect+steroid+hormone+synthesis+in+follicular+granulosa+cells%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">H3K9me3 involved in FOXL2 regulation of SerpinE2 expression to affect steroid hormone synthesis in follicular granulosa cells.</a> FASEB J. 39 (12), e70730(2025).</li>
<li>Panday, A., Elango, R., Willis, N. A., Scully, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modified+CUT%26RUN-seq+technique+for+qPCR+analysis+of+chromatin-protein+interactions%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">A modified CUT&RUN-seq technique for qPCR analysis of chromatin-protein interactions.</a> STAR Protoc. 3 (3), 101529(2022).</li>
<li>Lardo, S. M., Hainer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+factor+localization+on+chromatin+using+ultra-low+input+cleavage+under+targets+and+release+using+nuclease%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Single-cell factor localization on chromatin using ultra-low input cleavage under targets and release using nuclease.</a> J Vis Exp. (180), e63536(2022).</li>
<li>Hsieh, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resolving+the+3D+landscape+of+transcription-linked+mammalian+chromatin+folding%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">Resolving the 3D landscape of transcription-linked mammalian chromatin folding.</a> Mol Cell. 78 (3), 539-553.e8 (2020).</li>
<li>Mumbach, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HiChIP%3A+Efficient+and+sensitive+analysis+of+protein-directed+genome+architecture%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">HiChIP: Efficient and sensitive analysis of protein-directed genome architecture.</a> Nat Methods. 13 (11), 919-922 (2016).</li>
</ol>

The authors declare no competing interests.

Epigenetic dysregulation and LDTFs are key drivers of SCLC tumor biology6. Determining the genomic distribution of specific histone modifications and DNA-binding proteins in SCLC can reveal potential mechanisms of acquired therapeutic resistance and phenotypic plasticity. CUT&#38;RUN-seq is a powerful method for mapping histone and non-histone protein-DNA interactions at high resolution14 from cells, tissues. This protocol presents an effective and low-cost alternative to t...

Histone post-translational modifications (PTMs) and transcription factors (TFs) are central regulators of gene expression. Histone PTMs, such as acetylation, methylation, phosphorylation, and ubiquitination, primarily on their N-terminal tails influence chromatin structure1,2. In cancer, histone PTMs are often dysregulated, leading to aberrant activation of oncogenes or silencing of tumor suppressor genes3. TFs bind specific DNA sequences at promoters or enhancers to activate or repress target genes, often recruiting histone-modifying enzymes that coordinate chromatin remodeling4. Enzymes that write, erase, or read histone marks, such as histone acetyltransferases (HATs), histone deacetylases (HDACs), and methyltransferases (HMTs), are frequently mutated or dysregulated in cancer, making them attractive therapeutic targets5.
In SCLC, epigenetic changes and lineage-defining transcription factors (LDTFs) are key drivers of tumor biology6. Unlike genetic mutations, epigenetic alterations, including DNA methylation and histone modifications, regulate gene expression without changing the underlying DNA sequence. These modifications can silence tumor suppressor genes or activate oncogenes, fueling SCLC&#39;s aggressive behavior. For example, the histone methyltransferase KMT2D is frequently mutated with an alteration frequency of 12.9% in a large real-world SCLC patient cohort7 and is responsible for histone H3 lysine 4 mono-methylation (H3K4me1), a mark associated with active enhancer regions in the genome. SCLC is a molecularly heterogeneous disease. SCLC are classified into molecular subtypes based on differential expression of four LDTFs: achaete-scute homologue 1 (ASCL1), neurogenic differentiation factor 1 (NEUROD1), yes-associated protein 1 (YAP1) and POU class 2 homeobox 3 (POU2F3). The neuroendocrine (NE) subtypes, including ASCL1 and NEUROD1, account for approximately 70-80% of SCLC cases8. Therefore, investigating the distribution and profiles of different histone PTMs and TF binding sites in the various SCLC subtypes may elucidate subtype-specific gene programs vulnerable to therapeutic intervention.
The traditional method for identifying histone PTMs and the binding sites of sequence-specific regulatory proteins at the genome-wide level is chromatin immunoprecipitation sequencing (ChIP-seq)9. However, ChIP-seq requires a large number of input cells and often yields high background across the genome. As a result, ChIP-seq requires a high level of enrichment of target protein to distinguish true signal from noise and needs deep sequencing for effective data analysis. Additionally, formaldehyde crosslinking used in ChIP-seq can mask epitopes and generate false-positive binding sites10. Cleavage Under Targets and Release Using Nuclease followed by next-generation sequencing (CUT&#38;RUN-seq) is a high-resolution alternative for mapping histone PTMs and TF binding sites in situ within intact cells. In this method, permeabilized cells are incubated with an antibody targeting the chromatin-associated protein of interest, followed by binding of a Protein A(G)-micrococcal nuclease (MNase) fusion protein. Targeted DNA digestion flanking the protein of interest is then induced under high-calcium/low-salt conditions, releasing specific DNA fragments for purification. The purified DNA fragments are used to construct barcoded sequencing libraries, which can be pooled for high-throughput sequencing11,12,13,14. Compared to ChIP-seq, CUT&#38;RUN requires significantly fewer cells as input, produces lower background noise, avoids crosslinking artifacts, and requires fewer sequencing reads - making it particularly suitable for studying dynamic chromatin states in cancer in a cost-efficient manner.
CUT&#38;RUN technology has been developed to investigate the genomic locations of the chromatin-interacting proteins, including histone modifications, TF binding sites, and chromatin-associated complexes, thereby revealing epigenetic regulatory landscapes11,12. This technique has enabled detailed analyses of enhancer-promoter architecture, transcriptional network regulation, and chromatin state dynamics during development and disease progression15. In cancer research, CUT&#38;RUN allows precise mapping of histone marks16,17 and oncogenic transcription factor occupancy, helping to identify lineage-specific regulatory programs and potential therapeutic targets15,16. Furthermore, CUT&#38;RUN can be performed in situ in cells, including CD8+ T cell17, as well as to tissue samples18,19.
To detect the genomic location of PTMs and transcription factor E2F7 binding sites in SCLC subtypes, we applied CUT&#38;RUN-seq to four SCLC NE cell line models: H146 and DMS79 (ASCL1 subtype), as well as H446 and H82 (NEUROD1 subtype), with or without KMT2D mutations. We profiled the histone mark H3K4me1, an enhancer-associated modification primarily catalyzed by KMT2D, and E2F7, which has been shown to be a cofactor of ASCL120. The histone mark H3K4me3, which is associated with active promoters, served as positive control, while IgG served as negative control.
Here, we present a detailed CUT&#38;RUN-seq protocol for SCLC NE cell lines, based on the following: a commercially available CUT&#38;RUN kit (see Table of Materials), the original work of Skene and Henikoff11,12,13, and our own laboratory optimizations.

<table><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI-1640 media&amp;nbsp;</td><td>Gibco ThermoFisher Scientific</td><td>11875093</td><td>Cell culture medium</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Neuromics</td><td>FBS002-HI</td><td>Component of cell culture medium</td></tr><tr><td>1% Penicillin-Sstreptomycin&amp;nbsp;</td><td>Gibco ThermoFisher Scientific</td><td>15140122</td><td>Antibiotics,to prevent bacterial contamination of cell cultures</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Gibco ThermoFisher Scientific</td><td>15250061</td><td>Trypan Blue Staining was used to evaluate permeability with an automated cell counter.&amp;nbsp;</td></tr><tr><td>Roche cOmplete, EDTA-free Proease Inhibitor Cocktail</td><td>Roche</td><td>11873580001</td><td>Protease inhibitor. Compoents of wash buffer</td></tr><tr><td>5% Digitonin&amp;nbsp;</td><td>EpiCypher</td><td>21-1004</td><td>Buffer components&amp;nbsp; and Buffer recipe can be found in the Buffer sheet</td></tr><tr><td>Beads activation buffer</td><td>EpiCypher</td><td>21-1001</td><td>Buffer components&amp;nbsp; and Buffer recipe can be found in the Buffer sheet</td></tr><tr><td>Concanavalin A (ConA) Beads</td><td>EpiCypher</td><td>21-1401</td><td>ConA-conjugated paramagnetic microspheres are lectin-coated magnetic beads that permit lipid membrane binding</td></tr><tr><td>CUT&amp;amp;RUN Kit&amp;nbsp;</td><td>CUTANA ChIC EpiCypher</td><td>14-1048</td><td>Reagents for CUT&amp;amp;RUN DNA generation</td></tr><tr><td>DNA Purification Kit</td><td>CUTANA&amp;nbsp; EpiCypher</td><td>14-0050</td><td>Extract DNA from the released targeted DNA fragments in the supernantant in the reactIon</td></tr><tr><td>CUT&amp;amp;RUN Library Prep Kit</td><td>CUTANA EpiCypher</td><td>14-001</td><td>Generate the CUT&amp;amp;RUN next-generation sequencing (NGS) libraries</td></tr><tr><td>&amp;nbsp;DNA purification Magnetic Beads</td><td>Agencourt AMPure XP Beckman Coulter</td><td>A63880</td><td>PCR produt cleanup and size selection (i.e. adapter dimer and primer removal)</td></tr><tr><td>E. coli Spike-in DNA</td><td>EpiCypher</td><td>18-1401</td><td>Used as an exogenous spike-in to calibrate sequencing reads across a series of samples&amp;mdash;for example, treated versus untreated conditions</td></tr><tr><td>pAG-MNase</td><td>EpiCypher</td><td>&amp;nbsp;15-1016</td><td>Upon binding to the primary antibody, the enzyme cleaves the targeted DNA fragment on both sides of the bound protein when activated by calcium</td></tr><tr><td>PBS PH7.4 1x</td><td>Gibco (Thermo Fisher Scientific)</td><td>10010-049</td><td>Washing cells</td></tr><tr><td>Pre-Wash Buffer</td><td>EpiCypher</td><td>21-1002</td><td>Buffer components&amp;nbsp; and Buffer recipe can be found in the Buffer sheet</td></tr><tr><td>dsDNA High Sensitivity&amp;nbsp; Assay Kit</td><td>QubitTM Thermo Fisher Scientific</td><td>Q32854</td><td>CUT&amp;amp;RUN DNA quantification</td></tr><tr><td>Stop Buffer</td><td>EpiCypher</td><td>21-1003</td><td>Buffer components&amp;nbsp; and Buffer recipe can be found in the Buffer sheet</td></tr><tr><td>High Sensitivity DNA Kit&amp;nbsp;</td><td>Agilent Technoligies</td><td>5067-4626</td><td>CUT&amp;amp;RUN DNA and NGS libraies quatification and fragment size distribution analysis</td></tr><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>8-strip 0.2 mL PCR tubes</td><td>EpiCypher</td><td>10-0009k</td><td>Tubes for CUT&amp;amp;RUN reactions,compatible with the 0.2 ml tubes Magnetic Separation Rack</td></tr><tr><td>LUNA 8-channel slides</td><td>Logos BioSystems INC</td><td>L72001</td><td>Slides for cell counting,&amp;nbsp; cell viability and integrity checking</td></tr><tr><td>Multi-channel reagent reservoir</td><td>Fisher Scientific</td><td>14-387-072</td><td>Washing buffer container</td></tr><tr><td>DynaMagTM-2 (Magnetic separation rack) , 1.5 mL tubes</td><td>Thermo Fisher Scientific</td><td>12321D</td><td>ConA beads were isolated from their storage buffer by magnetic separation rack and subsequently activated by resuspension in activation buffer in a 1.5 mL microcentrifuge tube.</td></tr><tr><td>Magnetic Separation Rack, 0.2 mL tubes</td><td>EpiCypher</td><td>10-0008</td><td>Stand to separate&amp;nbsp; beads-binding cells, antibodies, pAG/MNase from reaction and washing buffers</td></tr><tr><td>Eppendor Research plus 8-Channel Pipette, 0.5-10 uL&amp;nbsp;</td><td>Eppendorf</td><td>3125000010</td><td>Aspiration 8-strip tubes&#039; samples&amp;nbsp; to accelerate workflow, comparable multi-channel pipettor</td></tr><tr><td>Eppendorf Refrence 2 8-Channel Pipette, 30-300 uL</td><td>Eppendorf</td><td>4926000050</td><td>Aspiration and wash steps to accelerate workflow, comparable multi-channel pipettor</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Scientific Industries Vortex-Genie 2 Mixer, Variable speed, 120V</td><td>Fisher Scientific</td><td>50-728-002</td><td>Min-centrifuge and spin</td></tr><tr><td>LUNA FX7 Automatic cell counter</td><td>Logos BioSystems INC</td><td>L70001</td><td>Cell Counter</td></tr><tr><td>Nutating Mixer</td><td>VWR</td><td>82007-202</td><td>Beads incubation steps (overnight antibody incubation, pAG-MNase digest reaction).</td></tr><tr><td>Eppendorf 6331 Nexus Gradient MasterCycler Thermal Cycler</td><td>Eppendorf</td><td>6331</td><td>CUT&amp;amp;RUN next generation library construction</td></tr><tr><td>Qubit 4 Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q33226</td><td>DNA quantification.</td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2939A</td><td>Fragment size, concentration analysis for CUT&amp;amp;RUN DNA and CUT&amp;amp;RUN libraries,&amp;nbsp; comparable capillary electrophoresis instrument (e.g. Agilent TapeStation)</td></tr><tr><td>Agilent TapeStation</td><td>Agilent Technologies</td><td>G2992AA</td><td>Fragment size, concentration analysis for CUT&amp;amp;RUN DNA and CUT&amp;amp;RUN libraries</td></tr><tr><td>Illumina NovaSeq 6000 system</td><td>&amp;nbsp;Illumina, San Diego, CA, USA</td><td></td><td>Sequencing the pooled CUT&amp;amp;RUN libraries&amp;nbsp;</td></tr></tbody></table>

genome-wide mapping of histone modifications and transcription factor binding sites in neuroendocrine small cell lung cancer cell lines using cut&amp;run

Genome-Wide Mapping of Histone Modifications and Transcription Factor Binding Sites in Neuroendocrine Small Cell Lung Cancer Cell Lines Using CUT&amp;RUN

Chromatin remodeling proteins and transcription factors (TFs) play critical roles in the tumor biology of small cell lung cancer (SCLC). Genome-wide characterization of histone post-translational modifications (PTMs) and TF binding sites is essential for identifying regulatory DNA elements and gene pathways that will lead to a deeper mechanistic understanding of SCLC and nominate targets for therapeutic intervention. Cleavage Under Targets and Release Using Nuclease followed by next generation sequencing (CUT&#38;RUN-seq) is a powerful method for mapping specific histone modifications and determining the DNA-binding profiles of a wide range of proteins in situ in the cellular genome. In CUT&#38;RUN, the micrococcal nuclease (MNase) fused to Protein A/G is recruited via antibodies to the genomic locations of chromatin-associated proteins, where the underlying DNA fragments are released from bulk chromatin upon MNase activation and cleavage. This localized digestion generates small, locus-specific DNA fragments suitable for sequencing. 
Here, we present a detailed protocol for profiling histone modifications H3K4me3 (associated with active or open promoters) and H3K4me1 (associated with active enhancers), as well as the transcription factor E2F7, in SCLC. This protocol has been optimized for neuroendocrine (NE) SCLC cell line models, which are typically characterized by large nuclei, scant cytoplasm, and growth as non-adherent aggregates in suspension.

Typical or "classic" SCLC NE cell lines are characterized by large nuclei, scant cytoplasm, and growth as non-adherent aggregates or compact spheroids in suspension. ASCL1-high cell lines, such as NCI-H146 and DMS79, and NEUROD1-high cell lines, such as NCI-H82 and H446, exhibit this classic SCLC phenotype: small-to-medium sized cells with scant cytoplasm, finely granular chromatin, and a tendency to grow predominantly as non-adherent aggregates or compact spheroids. Although NEUROD1-high models retain NE marker expressi...

An optimized Cleavage Under Targets and Release Using Nuclease followed by next generation sequencing (CUT&#38;RUN-seq) protocol is described for neuroendocrine small cell lung cancer cell lines. It enables genome-wide mapping of various histone modifications and transcription factor (e.g. E2F7) binding sites to investigate epigenetic and transcriptional deregulation in SCLC pathobiology.

Chromatin remodeling proteins and transcription factors (TFs) play critical roles in the tumor biology of small cell lung cancer (SCLC). Genome-wide ...

So the goal of our research is to identify the interactions between lineage defining transcription factors and chromatin remodeling proteins in the different types of small cell lung cancer, in regulating the transcription landscapes, and how these interactions can contribute to disease processes, including cancer initiation and progression. We aim to achieve this by conducting experiments like CUT&RUN, which is a next generation sequencing technique that can profile or identify at a global scale the genomic locations of where these differentially modified histones and DNA-bound transcription factors. Ultimately, we hope to identify new pathways that are epigenetically regulated that may be therapeutically actionable in the development of new treatments for patients with this very aggressive form of lung cancer.To begin, remove the cultured small cell lung cancer neuroendocrine cells from the 37 degrees Celsius incubator and examine them under a microscope to ensure their quality. Then transfer the cell suspension to a 50 milliliter conical tube and centrifuge the tube at 1, 200 g for five minutes at room temperature. Aspirate the medium from the conical tube by using the vacuum in the hood and rinse the resulting cell pellet with 20 milliliters of PBS.Centrifuge the tube again at 1, 200 g for five minutes, and aspirate the PBS. Add Accutase based on pellet size and incubate for three to five minutes at 37 degrees Celsius to digest the cell pellet. Next, terminate the digestion by adding five milliliters of PBS with 10%FBS and mixing to obtain a single cell suspension.After centrifuging the cells again as demonstrated earlier, resuspend the pellet in five milliliters of PBS. Count the starting cells and confirm their viability and integrity using the automatic cell counter. Calculate the total number of cells required and add 20%excess to account for pipetting errors.Transfer the calculated total number of cells to 1.5 milliliter tubes and centrifuge the tubes at 600 g for three minutes at room temperature. resuspend the cells in wash buffer at a density of five times 10 to the power of five cells per reaction for a total of six reactions. Use 100 microliters of buffer per reaction and pipette gently but thoroughly to ensure a uniform suspension.Centrifuge the cells again, remove the supernatant, and repeat the wash. Aliquot 100 microliters of the washed cells into each tube of an eight strip set that contains 10 microliters of activated Concanavalin A beads. Gently pipette to mix the contents.Incubate the bead cell slurry for 10 minutes at room temperature to allow cell binding to the beads, and quickly spin the tubes in a mini centrifuge to collect the slurry without allowing the beads to settle. Place the tubes on a 0.2 milliliter tube magnet rack. Allow the slurry to clear and pipette to remove the supernatant.Then remove the tubes from the magnet. Immediately add 50 microliters of ice cold antibody buffer to each reaction and gently pipette to resuspend the beads. Label the eight strip tubes appropriately.Add 0.5 micrograms each of H3K4me3, H3K4me1, and immunoglobulin G antibodies and 0.6 micrograms of E2F7 polyclonal antibody from different sources to each reaction tube. Gently pipette to mix the contents of each reaction tube thoroughly and incubate them overnight on a nutator at four degrees Celsius with tube caps elevated. Remove the eight strip tubes from the nutator and perform a quick spin to collect liquid at the bottom of each tube.Now place the tubes on the magnet stand until the slurry clears. Then carefully pipette to remove and discard the supernatant. Keeping the tubes on the magnet stand, wash each tube twice with 200 microliters of cold cell permeabilization buffer and use a multiple channel pipette to remove the supernatant after each wash.Next, remove the tubes from the magnet stand. Immediately add 50 microliters of cold cell permeable buffer to each reaction and gently pipette to resuspend the cells and beads. Then add 2.5 microliters of protein AG-micrococcal nuclease to each reaction and mix well by gently pipetting.After a 10 minute incubation at room temperature, perform a quick spin of the tubes. Place them back on the magnet stand until the slurry clears, and then remove the supernatant. After washing the beads twice with cold cell permeabilization buffer, gently resuspend them in 50 microliters of the cell permeable buffer.While keeping the tubes on ice, add one microliter of 100 millimolar calcium chloride to each reaction in the eight tube strip from the previous step. Gently pipette to fully resuspend the beads and promote efficient digestion. Incubate the tubes on a nutator at four degrees Celsius for two hours, keeping the tube caps elevated throughout the incubation.Now in a new 1.5 milliliter tube, prepare the stop buffer mix by combining 33 microliters of stop buffer with one microliter of escherichia coli spike in DNA at 0.5 nanograms per reaction. Gently vortex to mix the solution. At the end of the two hour incubation, add 33 microliters of the prepared stop buffer mixture to each reaction tube and mix thoroughly.Incubate the reaction tubes in a thermocycler at 37 degrees Celsius for 10 minutes. After a quick spin, place the tubes on a magnet rack until the solution becomes clear. Carefully transfer approximately 85 microliters of the supernatant containing the CUT&RUN release DNA into fresh 1.5 milliliter tubes.Purify the recovered DNA, assess its quantity, and perform fragment size distribution for quality control. Then proceed with CUT&RUN library preparation, quantification, indexed library pooling, and paired-end next generation sequencing. Achaete-scute homolog-1 high cell lines, DMS79 and H146, and Neurogenic Differentiation Factor 1 high cell lines, H82 grew as non-adherent aggregates or compact spheroids in suspension, while H446 exhibited mixed morphology, with approximately 80%inherent cells and 20%in suspension.CUT&RUN DNA fragment analysis revealed that the enhancer histone mark H3K4me1 yielded the highest DNA amounts across all cell lines, followed by H3K4me3, with E2F7 and IgG yielding the lowest amounts. For extracted CUT&RUN DNA, the fragment size distribution for H3K4me1 and H3K4me3 showed clear peaks for mono-nucleosome and di-nucleosome along with genomic DNA. In contrast, no distinct peaks were detected for E2F7 or the IgG control.Despite low DNA input, next generation sequencing libraries were successfully constructed for all CUT&RUN targets, including E2F7 and IgG. Heat map analysis in DMS79 wild type and H146 heterozygous mutant cells showed that H3K4me3 and E2F7 signals were enriched near transcription start sites, whereas H3K4me1 signals were more broadly distributed. Genome browser visualization at a randomly selected chromatin region showed distinct peaks for H3K4me3, H3K4me1, and E2F7, while IgG showed no enrichment.And so traditionally, scientists and researchers have used chromatin immunoprecipitation followed by sequencing or ChIP-seq to address questions like the ones that we are asking. However, ChIP-seq requires a rather high amount of input materials, which is not always feasible, depending on which model is being used. CUT&RUN, on the other hand, requires significantly less input material, as low as 50, 000 cells, and requires less high throughput sequencing reads, which means it's less costly, and produces far less background when it comes to data generation.CUT&RUN is a technique that can be applied to not only cells in 2D culture, but it can be adapted for other preclinical models, such as patient derived organoids and xenografts. It can also be used to profile the chromatin landscape in tissues, including both normal and diseased tissue. So this is an incredibly useful technology or technique for cancer researchers, but can be very useful as well for non-cancer researchers that are focused on studying different human diseases.This protocol allows a mapping of histone modifications and the abiding profiles of the diverse proteins in situ in the cellular genome. Providing histone PTMs and the TF binding across SCLC subtypes may reveal subtype specific targetable genes and regulatory elements, such as enhancers and the super-enhancers.

genome-wide-mapping-histone-modifications-transcription-factor

Research

JoVE Journal

Cancer Research

Unlike DNA sequence data, epigenomic data are not readily subjected to text-based searches. Presented here are the procedures to use an upgraded version of GeNemo, a web-based bioinformatics tool, to conduct pattern-based searches for similarities in epigenomic data comparing available online databases including Encyclopedia of DNA Elements with user's data.

Pattern-based Search of Epigenomic Data Using GeNemo

Here, we describe an optimized high-throughput ChIP-sequencing protocol and computational analyses pipeline for the determination of genome-wide chromatin state patterns from frozen tumor tissues and cell lines.

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

CUT&amp;RUN and its variants can be used to determine protein occupancy on chromatin. This protocol describes how to determine protein localization on chromatin using single-cell uliCUT&amp;RUN.

Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease

The increase of molecular biomarkers to be tested for non-squamous non-small cell lung cancer (NS-NSCLC) care management has prompted the development of fast and reliable molecular detection methods. We describe a workflow for genomic alteration assessment for NS-NSCLC patients using an ultra-fast-next generation sequencing (NGS) approach.

Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer

Researchers new to the epigenetic field will find CUT&amp;Tag a significantly easier alternative to ChIP assays. CUT&amp;Tag has tremendously benefited the epigenetic studies on rare and primary cell populations, generating high-quality data from very few cells. This protocol describes performing H3K4me1 CUT&amp;Tag assays on mouse myoblasts isolated from mouse hindlimb muscles.

Using Cleavage Under Targets and Tagmentation (CUT&#38;Tag) Assay in Mouse Myoblast Research

This study presents a spatial Trekker–CUT&amp;Tag multiome protocol combining direct spatial barcoding of a tissue section with single-cell multiome analysis. This approach enables spatially resolved single-cell profiling of gene expression and the histone H3K27ac modification simultaneously, offering mechanistic insights into gene regulation in the context of tissue microanatomy.

Spatially Resolved, Integrated Single-Cell Multiomic Profiling of the Transcriptome and Epigenomic Targets in Frozen Tissue Sections

Discover CUT&RUN-seq for genome-wide mapping of histone modifications and transcription factor binding in small cell lung cancer cell lines. This low-input method reveals epigenetic regulatory elements and potential therapeutic targets.

Genome-Wide Mapping of Histone Modifications and Transcription Factor Binding Sites in Neuroendocrine Small Cell Lung Cancer Cell Lines Using CUT and RUN (Video) | JoVE Journal

Genome-Wide Mapping of Histone Modifications and Transcription Factor Binding Sites in Neuroendocrine Small Cell Lung Cancer Cell Lines Using CUT&RUN

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