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

Summary

An optimized Cleavage Under Targets and Release Using Nuclease followed by next generation sequencing (CUT&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.

Abstract

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&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&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.

Introduction

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 structure^1,2. 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'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&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 sequencing^11,12,13,14. Compared to ChIP-seq, CUT&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&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 landscapes^11,12. 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&RUN allows precise mapping of histone marks^16,17 and oncogenic transcription factor occupancy, helping to identify lineage-specific regulatory programs and potential therapeutic targets^15,16. Furthermore, CUT&RUN can be performed in situ in cells, including CD8+ T cell¹⁷, as well as to tissue samples^18,19.

To detect the genomic location of PTMs and transcription factor E2F7 binding sites in SCLC subtypes, we applied CUT&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&RUN-seq protocol for SCLC NE cell lines, based on the following: a commercially available CUT&RUN kit (see Table of Materials), the original work of Skene and Henikoff^11,12,13, and our own laboratory optimizations.

Protocol

NOTE: This protocol (Figure 1) provides step-by-step instructions for performing CUT&RUN-seq in SCLC NE cell lines, which typically grow as non-adherent aggregates or floating clusters.

1. Cell Culture

1. 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 °C with 5% CO₂.
   NOTE: All cells used in this study were purchased from ATCC and have been authenticated with short tandem repeat profiling.

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&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 "dead") should be used for the CUT&RUN protocol.

1. Place the 5% digitonin stock, protease inhibitor, and spermidine on ice and allow them to thaw completely.
2. Prepare the Wash Buffer according to Table 1 and keep the buffer on ice .
3. Prepare a 0.05% digitonin working solution by mixing 10 µL of the 5% stock with 990 µL of Wash Buffer. Mix thoroughly by vortexing and keep on ice.
4. Generate a dilution series of digitonin (0.05%, 0.01%, 0.001%, and 0.0001%)”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 µL 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.
5. Keep all digitonin dilution solutions and the DMSO control on ice until they are used.
6. 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 5×10⁵ cells each, prepare approximately 1.2 x 5 x 5 x 10⁵ cells).
7. Collect the required number of cultured cells by centrifugation at 600 × g for 3 min at room temperature (RT). Resuspend the pellet in 600 µL of 1× PBS (100 µL per condition plus 20% extra).
8. Aliquot 100 µL of the PBS-resuspended cells into five 1.5 mL microcentrifuge tubes- one tube for each digitonin concentration and one for the DMSO control.
9. Centrifuge the tubes at 600 × g for 3 min at RT.
10. Carefully remove the supernatant and resuspend each pellet in 50 µL of the corresponding digitonin or DMSO control buffer.
11. Incubate the samples at RT for 10 min.
12. Mix 10 µL of the treated cells with 10 µL of trypan blue and load 10 uL of the mixture onto a cell counting slide.
13. Use a cell counter to quantify viable cells (unstained) and non-viable, permeabilized positive cells (stained blue).
14. Identify the lowest digitonin concentration that produces the highest proportion of trypan blue positive cells. Use this concentration for the downstream CUT&RUN experiment. Confirm that the DMSO control maintains viability typical of a normal healthy cell culture.

3. CUT&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&RUN experiment.

1. Day 1
   1. CUT&RUN buffer prep
      1. Prepare the Wash buffer by combining Pre-Wash Buffer, 25x Protease Inhibitor, and 1 M Spermidine as outlined in Table 3.
      2. Prepare the Cell Permeabilization Buffer by adding 5% Digitonin to the Wash Buffer according to Table 3.
      3. Prepare the Antibody Buffer by adding 0.4 µL of EDTA to 100 µL of Cell Permeabilization Buffer with the appropriate digitonin concentration, as described in Table 3. Place the prepared buffer on ice.
      4. Store the remaining Cell Permeabilization Buffer at 4 °C for use on Day 2.
   2. Concanavalin A (ConA) beads activation
      1. Gently resuspend ConA beads suspension to ensure its even distribution.
      2. Transfer 55 µL of the beads suspension (11 µL per sample) into a 1.5 mL microcentrifuge tube.
      3. Place tube on a 1.5 mL magnetic rack until the beads have fully separated, then pipette to remove supernatant.
      4. Immediately wash the beads twice with 500 µL of ice-cold Bead Activation Buffer (100 µL per sample). Resuspend the beads by gentle pipetting for each wash and use the magnetic rack to discard supernatant between washes.
      5. After the final wash, remove the supernatant and gently resuspend the activated beads in 55 µL (11 µL per sample) of ice-cold Bead Activation Buffer.
      6. Aliquot 10 µL of the beads suspension into each tube of an 8-strip tube set and keep the tubes on ice.
   3. 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.
      1. Remove the cells from the 37 °C incubator and examine them under a microscope to ensure quality.
      2. Transfer the cell suspension to a 50 mL conical tube and centrifuge at 1,000-1,200 × g for 5 min at RT.
      3. Aspirate the medium from the conical tube and rinse the cell pellet with 20 mL of 1× PBS.
      4. Centrifuge again, aspirate the PBS, and digest the pellet with Accutase for 3-5 min at 37°C, based on the size of the pellet.
      5. 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.
      6. Centrifuge the cells at 1,000-1,200 × g for 5 min, discard the supernatant, and resuspend the cell pellet in 5 mL of PBS.
      7. Count starting cells, confirm cell viability and integrity.
      8. Calculate the total number of cells needed by multiplying the number of reactions by the number of cells per reaction (5×10⁵). Add 20% excess to account for pipetting errors (e.g., 1.2 × 6 × 5 × 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.
      9. Resuspend the cells in RT Wash Buffer at a density of 5×10⁵ cells per reaction (six reactions total), using 100 µL 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.
      10. Repeat the wash step (Step 9) once more.
      11. Aliquot 100 µL of washed cells to each tube in the 8-strip set containing 10 µL of activated CoA beads. Gently pipette to mix and quickly spin in a mini centrifuge to collect the slurry (beads should not settle).
      12. Incubate bead-cell slurry for 10 min at RT to allow cells to bind to the beads.
      13. Place tubes on a 0.2 mL tubes magnet rack, allow the slurry to clear, and pipette to remove supernatant. Save 10 µL of supernatant to confirm cells are bound to beads. Discard the remaining supernatant and move quickly to the next step.
      14. Remove tubes from the magnet. Immediately add 50 µL of ice-cold Antibody Buffer to each reaction and gently pipette to resuspend the beads.
      15. Mix 10 µL of both the supernatant and 10 µL of the cell/bead suspension (resuspended in Antibody Buffer) with 10 µL of trypan blue and load the mixture onto the cell counter slide.
      16. 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.
   4. Permeabilization and antibody binding
      NOTE: The success of CUT&RUN critically depends on the antibody'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&RUN when available. 2. For targets without CUT&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&RUN. 3. Test 3-5 antibodies in parallel to identify the most effective one for your specific application.
      1. Quick spin the K-MetStat Panel and pipette to resuspend. Add 2 µL of K-MetStat Panel to reactions designated for H3K4me3 positive & IgG negative control antibodies. Pipette to mix and quick spin tubes (Optional).
      2. Add 0.5 µg each of H3K4me3, H3K4me1, and IgG antibodies, and 0.6 µg of E2F7 polyclonal antibody from different sources to each reaction (Table 4). Label the 8-strip tubes.
      3. Gently pipette to properly mix each reaction tube. Incubate overnight at 4 °C on a nutator with the tube caps elevated. Do not rotate or invert the tubes^.
2. Day 2
   1. 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.
      1. Put the magnet stand on ice.
      2. Remove the 8-strip tubes from 4 ˚C incubation and quick spin to collect liquid.
      3. Place tubes on the magnet stand until the slurry clears. Carefully pipette and remove the supernatant.
      4. Keep tubes on the magnet stand. Wash tubes twice with 200 µL of cold Cell Permeabilization Buffer. Pipette to remove supernatant.
      5. Remove tubes from the magnet stand and immediately add 50 µL of cold Cell Permeabilization Buffer to each reaction. Gently pipetting to resuspend cells/beads.
      6. Add 2.5 µL of pAG-MNase to each reaction and mix well by gently pipetting.
      7. Incubate reactions for 10 min at RT.
      8. Quick spin the tubes, place them back on the magnet stand, until the slurry clears. Pipette to remove supernatant.
      9. Keeping tubes on magnet stand, wash tubes twice with 200 µL of cold Cell Permeabilization Buffer. Pipette to remove supernatant.
      10. Remove tubes from the magnet stand, gently resuspend in 50 µL of cold Cell Permeabilization Buffer. Place the tubes on ice.
   2. Targeted chromatin digestion and release
      NOTE: Perform Ca²⁺-dependent digestion at 0 °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.
      1. Add 1 µL 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.
      2. Incubate the tubes on a nutator at 4 °C for 2 h, with the capped ends elevated.
      3. In a new 1.5 mL tube, prepare the Stop Buffer Mix. For each reaction, combine 33 µL of Stop Buffer with 1 µL of E. coli spike-in DNA (0.5 ng). Gently vortex to mix.
      4. Add 33 µL of stop buffer to each reaction at the end of the 2 h incubation. Mix by gentle pipetting.
      5. Incubate the reactions in a thermocycler at 37 °C for 10 min.
      6. Quick spin tubes to collect the contents at the bottom, then place them on a magnet rack until the solution becomes clear.
      7. Carefully transfer approximately 84 µL supernatant which contains the CUT&RUN-released DNA into fresh 1.5 mL tubes.
   3. 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 ≥ 95% ethanol to the DNA Wash Buffer.
      1. Add 420 µL of DNA Binding Buffer to each collected supernatant. Vortex briefly to mix thoroughly.
      2. Place a DNA Cleanup column into its collection tubes.
      3. Load each sample onto the corresponding column.
      4. Centrifuge at 16,000 × g for 30 s at RT. Discard the flow-through and return the column to the collection tube.
      5. Add 200 µL 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.
      6. Repeat the washing step once.
      7. Centrifuge again at 16,000 x g for 30 s at RT to fully remove residual wash buffer.
      8. Transfer columns to labeled 1.5 mL tubes.
      9. Apply 12 µL of Elution Buffer directly to the membrane at the center of the column.
      10. Incubate for 5 min at RT and then centrifuge at 16,000 × g for 1 min at RT to elute the DNA.
          NOTE: This is a safe stopping point. Store eluted DNA at -20 ˚C for future processing.
   4. CUT&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.
      1. Use 1 µL of each sample to determine CUT&RUN DNA concentration using the dsDNA High Sensitivity Assay Kit (see Table of Materials).
      2. Use an additional 1 µL 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'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.
   5. Library construction, fragment analysis, and sequencing
      NOTE: ≤5 ng total CUT&RUN DNA, as determined by dsDNA High Sensitivity Assay Kit quantification, was used for CUT&RUN next-generation sequencing (NGS) libraries' construction. To minimize adapter dimers, reduce the Adapter from Illumina to 1 µL 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.
      1. Library construction was built by using the CUT&RUN library prep kit (see Table of Materials) according to the kit manufacturer's instructions, with small modifications. In brief, 5 ng of CUT&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 & 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˚C for further processing.
      2. Measure the concentration of CUT&RUN library with a Fluorometer and the dsDNA High Sensitivity Assay Kit. The size distribution of the CUT&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&RUN library to bring the concentration within the detection range of the dsDNA High Sensitivity Assay Kit and the High Sensitivity DNA Kit.
      3. 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.
   6. CUT&RUN data analysis
      1. Analyze CUT&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&RUN)¹⁹.

Results

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...

Discussion

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&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...

Materials

List of materials used in this article

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