We thank members of the Hainer Lab for reading and comments on an earlier version of this manuscript. This project used the NextSeq500 available at the University of Pittsburgh Health Sciences Sequencing Core at UPMC Children&#39;s Hospital of Pittsburgh for sequencing with special thanks to its director, William MacDonald. This research was supported in part by the University of Pittsburgh Center for Research Computing through the computer resources provided. This work was supported by the National Institutes of Health Grant Number R35GM133732 (to S.J.H.).

Ethics statement: All studies were approved by the Institutional Biosafety Office of Research Protections at the University of Pittsburgh.
1. Prepare magnetic beads
NOTE: Perform prior to cell sorting and hold on ice until use.
<ol>
	<li>Pipette 30 &#181;L of ConA-conjugated paramagnetic microspheres bead slurry mix per reaction to a fresh 1.5 mL microfuge tube and add 850 &#181;L of Binding Buffer, pipetting gently to mix. 
	NOTE: ConA-conjugated paramagnetic microspheres are lectin-coated magnetic beads that permit lipid membrane binding.</li>
	<li>Place the tube on a magnetic rack and allow the beads to magnetize for 1-2 min. Once the supernatant has cleared, remove&#160;and discard the supernatant without disturbing the beads.</li>
	<li>Remove the tube from the magnetic rack and wash the beads by resuspending in 1 mL of Binding Buffer.</li>
	<li>Repeat steps 1.2 and 1.3.</li>
	<li>Magnetize the beads for 2 min and remove the supernatant to discard.</li>
	<li>Remove the tube from the magnetic rack and resuspend the beads in 30 &#181;L of Binding Buffer per reaction.</li>
	<li>Hold the washed bead mix on ice until cells are sorted.</li>
</ol>
2. Harvest cells
NOTE: This step is written for adhered cells and optimized for murine E14 embryonic stem cells. Culturing and harvesting the cells depend on the cell type.
<ol>
	<li>Remove the cells from the 37 &#176;C incubator and examine them under a microscope to assure quality.</li>
	<li>Aspirate the media from the cell plate and rinse with 5 mL of 1x PBS.</li>
	<li>Aspirate PBS from the plate and harvest the cells (using traditional cell harvesting methods which will differ by cell type). Obtain single-cell suspension by gently pipetting up and down with a serological pipette against the culture dish, if necessary.</li>
	<li>Transfer the cell suspension to a 15 mL conical tube and spin down at 200 x g for 5 min.</li>
	<li>Aspirate off the media to discard and wash the cell pellet with 5 mL of PBS + 1% FBS.</li>
	<li>Spin down the cells at 200 x g for 5 min, discard the supernatant, and resuspend the cell pellet in 5 mL of PBS + 1% FBS.</li>
	<li>Count the cells and transfer 1 mL of 1 x 106 cells into a fresh 1.5 mL microfuge tube.</li>
	<li>Add 5 &#181;L of 7-Amino-Actinomycin D (7-AAD), invert the tube well to mix, and then apply the sample to the cell sorter to sort live single cells into individual wells of a 96-well plate. 
	NOTE: 7-AAD dye is excluded from live cells, and therefore can be used in live-cell sorting.</li>
</ol>
3. Cell sorting and lysis
<ol>
	<li>Prepare a cell sorter-compatible 96-well plate with 100 &#181;L of Nuclear Extraction (NE) Buffer in each well prior to cell sorting.</li>
	<li>Sort the cells into 96-well plates following the manufacturer&#39;s instructions.</li>
	<li>Quickly spin the plate (600 x g for 30 s) to assure cells are in the buffer within the wells. 
	NOTE: It is worth testing in a preliminary experiment whether the cells being used are reliably brought to the bottom of the wells.</li>
	<li>Hold the samples on ice for 15 min.</li>
	<li>Spin down the samples at 600 x g for 5 min at 4 &#176;C and carefully pipette to remove the supernatant (leaving behind 5 &#181;L).</li>
	<li>Resuspend each sample in 55 &#181;L of NE buffer and add 30 &#181;L of the prewashed ConA-conjugated paramagnetic microspheres (from step 1.7; in Binding Buffer) to each reaction.</li>
	<li>Incubate at room temperature for 10 min.</li>
</ol>
4. Pre-block samples to prevent early digestion by MNase
<ol>
	<li>Place the plate on a 96-well magnetic rack, allow the beads to bind for a minimum of 5 min, and then remove and discard the supernatant.</li>
	<li>Add 100 &#181;L of Blocking Buffer to the nucleus-bound beads and mix with gentle pipetting.</li>
	<li>Incubate for 5 min at room temperature.</li>
</ol>
5. Addition of primary antibody
<ol>
	<li>Place the plate on a 96-well magnetic rack, allow the supernatant to clear for a minimum of 5 min, and then remove and discard the supernatant without disturbing the beads.</li>
	<li>Remove the plate from the magnetic rack and resuspend the beads in 100 &#181;L of Wash Buffer per reaction with gentle pipetting.</li>
	<li>Place the plate back on the 96-well magnetic rack, allow the supernatant to clear, and then remove and discard the supernatant.</li>
	<li>Resuspend the beads in 25 &#181;L of Wash Buffer per reaction with gentle pipetting.</li>
	<li>Make a primary antibody master mix: 25 &#181;L of Wash Buffer + 0.5 &#181;L of antibody per reaction.</li>
	<li>While gently vortexing the nuclei-bound beads, add 25 &#181;L of the primary antibody master mix to each sample being treated with an antibody targeting the protein of interest (typically 1:100 final dilution). Add 25 &#181;L of Wash Buffer with no antibody, if performing a control.</li>
	<li>Incubate for 1 h at room temperature.</li>
	<li>Place the samples on a 96-well magnetic rack, allow the supernatant to clear for a minimum of 5 min, and then remove and discard the supernatant without disturbing the beads.</li>
	<li>Remove the plate from the magnetic rack and wash the beads with 100 &#181;L of Wash Buffer, resuspending by pipetting.</li>
</ol>
6. Addition of pA-MNase or pA/G-MNase
NOTE: Protein A has a high affinity for IgG molecules from certain species such as rabbits but is not suitable for IgGs from other species such as mice or rats. Alternatively, Protein A/G-MNase can be used. This hybrid binds rabbit, mouse, and rat IgGs, avoiding the need for secondary antibodies when mouse or rat primary antibodies are used.
<ol>
	<li>Place the plate back on a 96-well magnetic rack, allow the supernatant to clear for a minimum of 5 min, and then remove and discard the supernatant without disturbing the beads.</li>
	<li>Remove the plate from the magnetic rack and resuspend each sample in 25 &#181;L of Wash Buffer.</li>
	<li>Make a pA-MNase master mix (25 &#181;L of Wash Buffer + optimized amount of pA-MNase per reaction).</li>
	<li>While gently vortexing, add 25 &#181;L of the pA-MNase master mix to each sample, including the control samples. 
	NOTE: The concentration of pA-MNase varies upon preparation, if homemade, and should be&#160;tested prior to use upon each independent purification. For pA/G-MNase, 2.5 &#181;L of the 20x stock should be used.</li>
	<li>Incubate the samples for 30 min at room temperature.</li>
	<li>Place the plate on a 96-well magnetic rack, allow the supernatant to clear for a minimum of 5 min, and then remove and discard the supernatant without disturbing the beads.</li>
	<li>Remove the plate from the magnetic rack and wash the beads with 100 &#181;L of Wash Buffer, resuspending by gentle pipetting.</li>
</ol>
7. Directed DNA digestion
<ol>
	<li>Place the plate on a 96-well magnetic rack, allow the supernatant to clear for a minimum of 5 min, and then remove and discard the supernatant.</li>
	<li>Remove the samples from the magnetic rack and resuspend the beads in 50 &#181;L of Wash Buffer by gentle pipetting.</li>
	<li>Equilibrate the samples to 0 &#176;C in an ice/water mixture for 5 min.</li>
	<li>Remove the samples from the 0 &#176;C ice/water bath and add 1 &#181;L of 100 mM CaCl2 using a multichannel pipette. Mix well (3-5 times) with gentle pipetting using a larger volume multichannel pipette, and then return the samples to 0 &#176;C. 
	NOTE: Mixing well here is essential. CaCl2 is added to activate MNase digestion of DNA flanking the protein of interest.</li>
	<li>Start a 10 min timer as soon as the plate is back in the ice/water bath.</li>
	<li>Stop the reaction by pipetting 50 &#181;L of 2XRSTOP+ Buffer into each well, in the same order as the CaCl2 was added. 
	NOTE: Make 2XRSTOP+ Buffer before the 10 min digestion is over to prevent over digestion.</li>
</ol>
8. Sample fractionation
<ol>
	<li>Incubate the samples for 20 min at 37 &#176;C.</li>
	<li>Spin the plate at 16,000 x g for 5 min at 4 &#176;C.</li>
	<li>Place the plate on a 96-well magnetic rack, allow the supernatant to clear for a minimum of 5 min, and transfer supernatants to a fresh 96-well plate. Discard the beads.</li>
</ol>
9. DNA extraction
<ol>
	<li>Add 1 &#181;L of 10% Sodium Dodecyl Sulfate (SDS) and 0.83 &#181;L of 20 mg/mL Proteinase K to each sample. 
	CAUTION: SDS powder is harmful if inhaled. Users should use in well-ventilated spaces wearing goggles, gloves, and an N95-grade respirator, handling with care.</li>
	<li>Mix the samples by gentle pipetting.</li>
	<li>Incubate the samples for 10 min at 70 &#176;C.</li>
	<li>Return the plate to room temperature and add 46.6 &#181;L of 5 M NaCl and 90 &#181;L of 50% PEG 4000. Mix by gentle pipetting.</li>
	<li>Add 33 &#181;L of polystyrene-magnetite beads to each sample and incubate for 10 min at room temperature. 
	NOTE: Be sure to bring polystyrene-magnetite beads to room temperature (~30 min) and mix well before using.</li>
	<li>Place the plate on a magnetic rack and allow the supernatant to clear for ~5 min, and then carefully discard the supernatant without disturbing the beads.</li>
	<li>Rinse 2x with 150 &#181;L of 80% ethanol without disturbing the beads. 
	CAUTION: Ethanol is highly flammable and causes skin, eye, and lung irritation. Perform this step with appropriate laboratory clothing and in a vented hood.</li>
	<li>Spin the plate briefly at 1000 x g for 30 s. Place the plate back on a 96-well magnetic rack and remove all residual EtOH without disturbing the beads.</li>
	<li>Air-dry the samples for ~2-5 min. 
	NOTE: Do not dry the beads for longer than 5 min. If diligent about removing the EtOH, 2-3 min of drying is sufficient.</li>
	<li>Resuspend the beads with 37.5 &#181;L of 10 mM Tris-HCl (pH 8) and incubate for 5 min at room temperature.</li>
	<li>Place the plate on a magnetic rack&#160;and allow the beads to bind for 5 min.</li>
	<li>Transfer 36.5 &#181;L of the supernatant to a fresh thermocycler compatible 96-well plate. Discard the beads. 
	NOTE: The experiment can be stopped here by storing samples at -20 &#176;C or can continue with the library build (steps 10-15).</li>
</ol>
10. End repair, phosphorylation, adenylation
NOTE: The reagents are sourced as referenced in the Table of Materials. The below protocol follows a similar method to the commercial kit such as NEBNext Ultra DNA II kit.
<ol>
	<li>Dilute 5 U/&#181;L of T4 DNA polymerase 1:20 in 1x T4 DNA ligase buffer.</li>
	<li>Prepare an end-repair/3&#39;A master mix: 2 &#181;L of 10x T4 DNA ligase buffer, 2.5 &#181;L of 10 mM dNTPs, 1.25 &#181;L of 10 mM ATP, 3.13 &#181;L of 40% PEG 4000, 0.63 &#181;L of 10 U/&#181;L T4 PNK, 0.5 &#181;L of diluted T4 DNA polymerase, 0.5 &#181;L of 5U/&#181;L Taq DNA polymerase, with a total volume of 13.5 &#181;L per reaction. 
	NOTE: Be sure to bring 40% PEG 4000 to room temperature before pipetting.</li>
	<li>Add 13.5 &#181;L of end-repair/3&#39;A master mix to 36.5 &#181;L of DNA.</li>
	<li>Mix the reaction by quick vortex, and then a quick spin (500 x g for 10 s).</li>
	<li>Incubate using the following reaction conditions in a pre-cooled thermocycler with a heated lid for temperatures &#62;20 &#176;C. Use the reaction conditions: 12 &#176;C for 15 min, 37 &#176;C for 15 min, 72 &#176;C for 20 min, hold at 4 &#176;C.</li>
</ol>
11. Adapter ligation
NOTE: Keep the samples on ice while setting up the following reaction. Allow ligase buffer to come to room temperature before pipetting. Dilute the Adaptor (see Table of Materials) in a solution of 10 mM Tris-HCl containing 10 mM NaCl (pH 7.5). Due to the low yield, do not pre-quantify the CUT&#38;RUN-enriched DNA. Rather, generate 25-fold dilutions of the adaptor, using a final working adaptor concentration of 0.6 &#181;M.
<ol>
	<li>Make a ligation master mix: 55 &#181;L of ligase buffer (2x), 5 &#181;L of T4 DNA ligase, and 5 &#181;L of diluted Adaptor, with a total volume of 65 &#181;L per reaction.</li>
	<li>Add 65 &#181;L of the master ligation mix to 50 &#181;L of DNA from step 10.5.</li>
	<li>Mix by quick vortexing, followed by quick spinning (500 x g for 10 s).</li>
	<li>Incubate at 20 &#176;C in a thermocycler (without a heated lid) for 15 min. 
	NOTE: Proceed immediately to the following step.</li>
</ol>
12. USER digestion
<ol>
	<li>Add 3 &#181;L of USER enzyme to each sample, vortex, and spin (500 x g for 10 s).</li>
	<li>Incubate in thermal cycler at 37 &#176;C for 15 min (heated lid set to 50 &#176;C).</li>
</ol>
13. Polystyrene-magnetite bead clean-up following ligation reaction
NOTE: Allow polystyrene-magnetite beads to equilibrate at room temperature (~30 min). Vortex to homogenize the bead solution before using. Perform the following steps at room temperature.
<ol>
	<li>Add 39 &#181;L (0.33x) of polystyrene-magnetite bead solution to each well containing adaptor-ligated DNA.</li>
	<li>Mix thoroughly by pipetting, and then incubate the samples at room temperature for 15 min to allow DNA to bind to the beads.</li>
	<li>Place the samples on a 96-well magnetic rack and incubate for 5 min until the supernatant is clear.</li>
	<li>Keep the plate on the magnetic rack and carefully remove and discard the supernatant without disturbing the beads.</li>
	<li>Rinse the beads with 200 &#181;L of 80% EtOH without disturbing the beads.</li>
	<li>Incubate for 30 s on a 96-well magnetic rack to allow the solution to clear.</li>
	<li>Remove and discard the supernatant without disturbing the beads.</li>
	<li>Repeat steps 13.5-13.7 for a total of two washes.</li>
	<li>Spin the plate briefly at 500 x g for 10 s, place the plate back on a 96-well magnetic rack, and remove residual EtOH without disturbing the beads.</li>
	<li>Keep the plate on the magnetic rack and air-dry the samples for 2 min. 
	NOTE: Do not over-dry the beads.</li>
	<li>Remove the plate from the magnetic rack and resuspend the beads in 28.5 &#181;L of 10 mM Tris-HCl (pH 8). 
	CAUTION: Hydrochloric acid is very corrosive. Users should handle it with care in a chemical fume hood wearing goggles, gloves, and a lab coat.</li>
	<li>Thoroughly resuspend beads by pipetting, taking care to not produce bubbles.</li>
	<li>Incubate for 5 min at room temperature.</li>
	<li>Place the plate on a 96-well magnetic rack and allow the solution to clear for 5 min.</li>
	<li>Transfer 27.5 &#181;L of supernatant to a new PCR plate and discard the beads.</li>
</ol>
14. Library enrichment
NOTE: Primers are diluted with the same solution as the adaptor. For this library build, use a final working primer concentration of 0.6 &#181;M.
<ol>
	<li>Add 5 &#181;L of diluted indexed primer (see Table of Materials) to each sample. 
	NOTE: Each sample needs a different index to be identified after&#160;sequencing.</li>
	<li>Prepare a PCR master mix: 10 &#181;L of 5x high fidelity PCR buffer, 1.5 &#181;L of 10 mM dNTPs, 5 &#181;L of diluted Universal primer, 1 &#181;L of 1 U hot start high fidelity polymerase, with a total master mix volume of 17.5 &#181;L per sample.</li>
	<li>Add 17.5 &#181;L pf PCR mix to 32.5 &#181;L of purified adaptor-ligated DNA (5 &#181;L of indexed primer is included in this volume).</li>
	<li>Mix the solution by pipetting.</li>
	<li>Incubate in a thermocycler using the following reaction conditions with a maximum ramp rate of 3 &#176;C/s: 98 &#176;C for 45 s, 98 &#176;C for 45 s, 60 &#176;C for 10 s, repeat the second and third steps 21 times, 72 &#176;C for 1 min, hold at 4 &#176;C. 
	NOTE: The samples can be kept at 4 &#176;C for short-term storage or -20 &#176;C for long-term storage.</li>
</ol>
15. Polystyrene-magnetite bead clean-up
<ol>
	<li>Add 60 &#181;L (1.2x) of polystyrene-magnetite beads to each sample.</li>
	<li>Resuspend the beads by pipetting and incubate for 15 min at room temperature.</li>
	<li>Place the plate on a magnetic rack for 5 min until the solution is clear.</li>
	<li>Discard the supernatant and rinse the beads with 200 &#181;L of 80% EtOH without disturbing the beads.</li>
	<li>Repeat the wash step for a total of two 80% EtOH washes.</li>
	<li>Spin the plate at 500 x g for 10 s, place the plate on a 96-well magnetic rack, and allow the beads to bind for 5 min.</li>
	<li>Pipette to remove excess EtOH without disturbing the beads and allow the beads to air-dry for 2 min. 
	NOTE: Do not over-dry the beads.</li>
	<li>Resuspend the beads in 21 &#181;L of nuclease-free water and incubate for 5 min at room temperature.</li>
	<li>Place the plate on a magnetic rack and allow the solution to clear for 5 min.</li>
	<li>Transfer 20 &#181;L of the supernatant to a new plate. 
	NOTE: The experiment can be stopped here by storing the sample at -20 &#176;C.</li>
	<li>Quantify library concentrations with a fluorometer (see Table of Materials), using a 1x HS reagent.</li>
	<li>If the concentration allows, run 30 ng of sample on 1.5% agarose gel with a low molecular weight ladder to visualize. Alternatively, visualize on a Fragment Analyzer or related instrument.</li>
	<li>Sequence libraries on an Illumina platform to obtain ~50,000-100,000 uniquely mapped reads.</li>
</ol>

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J., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+targeted+nuclease+strategy+for+high-resolution+mapping+of+DNA+binding+sites.">An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites.</a> eLife. 6, 21856 (2017).</li><li>Meers, M. P., Bryson, T. D., Henikoff, J. G., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+CUT&amp;RUN+chromatin+profiling+tools.">Improved CUT&amp;RUN chromatin profiling tools.</a> eLife. 8, 46314 (2019).</li><li>Hainer, S. J., Bo&#353;kovi&#263;, A., McCannell, K. N., Rando, O. J., Fazzio, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+pluripotency+factors+in+single+cells+and+early+embryos.">Profiling of pluripotency factors in single cells and early embryos.</a> Cell. 177 (5), 1319-1329 (2019).</li><li>Kaya-Okur, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CUT&amp;Tag+for+efficient+epigenomic+profiling+of+small+samples+and+single+cells.">CUT&amp;Tag for efficient epigenomic profiling of small samples and single cells.</a> Nature Communications. 10 (1), 1930 (2019).</li><li>Carter, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+histone+modifications+in+low+cell+number+and+single+cells+using+antibody-guided+chromatin+tagmentation+(ACT-seq).">Mapping histone modifications in low cell number and single cells using antibody-guided chromatin tagmentation (ACT-seq).</a> Nature Communications. 10 (1), 3747 (2019).</li><li>Gopalan, S., Wang, Y., Harper, N. W., Garber, M., Fazzio, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+profiling+of+multiple+chromatin+proteins+in+the+same+cells.">Simultaneous profiling of multiple chromatin proteins in the same cells.</a> Molecular Cell. 81 (22), 4736-4746 (2021).</li><li>Patty, B. J., Hainer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+chromatin+profiling+genome-wide+using+uliCUT&amp;RUN+in+single+cells+and+individual+blastocysts.">Transcription factor chromatin profiling genome-wide using uliCUT&amp;RUN in single cells and individual blastocysts.</a> Nature Protocols. 16 (5), 2633-2666 (2021).</li><li>Zheng, X. -. Y., Gehring, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-input+chromatin+profiling+in+Arabidopsis+endosperm+using+CUT&amp;RUN.">Low-input chromatin profiling in Arabidopsis endosperm using CUT&amp;RUN.</a> Plant Reproduction. 32 (1), 63-75 (2019).</li><li>Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y., Greenleaf, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposition+of+native+chromatin+for+fast+and+sensitive+epigenomic+profiling+of+open+chromatin,+DNA-binding+proteins+and+nucleosome+position.">Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position.</a> Nature Methods. 10 (12), 1213-1218 (2013).</li><li>Buenrostro, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+chromatin+accessibility+reveals+principles+of+regulatory+variation.">Single-cell chromatin accessibility reveals principles of regulatory variation.</a> Nature. 523 (7561), 486-490 (2015).</li></ol>

The authors declare no competing interests related to this project.

CUT&#38;RUN is an effective protocol to determine protein localization on chromatin. It has many advantages relative to other protocols, including: 1) high signal-to-noise ratio, 2) rapid protocol, and 3) low sequencing read coverage required thus leading to cost savings. The use of Protein A- or Protein A/G-MNase enables CUT&#38;RUN to be applied with any available antibody; therefore, it has the potential to quickly and easily profile many proteins. However, adaptation to single-cell for any protein profiling on chroma...

Many nuclear proteins function by interacting with chromatin to promote or prevent DNA-templated activities. To determine the function of these chromatin-interacting proteins, it is important to identify the genomic locations at which these proteins are bound. Since its development in 1985, Chromatin Immunoprecipitation (ChIP) has been the gold standard for identifying where a protein binds to chromatin1,2. The traditional ChIP technique has the following basic workflow: cells are harvested and crosslinked (usually with formaldehyde), chromatin is sheared (usually with harsh sonication methods, necessitating crosslinking), the protein of interest is immunoprecipitated using an antibody that targets the protein (or tagged protein) followed by a secondary antibody (coupled to agarose or magnetic beads), crosslinking is reversed, protein and RNA are digested to purify DNA, and this ChIP enriched-DNA is used as the template for analysis (using radiolabeled probes1,2, qPCR3, microarrays5,6, or sequencing4). With the advent of microarrays and massively parallel deep sequencing, ChIP-chip5,6 and ChIP-seq4 have more recently been developed and allow for genome-wide identification of protein localization on chromatin. Crosslinking ChIP has been a powerful and reliable technique since its advent with major advances in resolution by ChIP-exo7 and ChIP-nexus8. In parallel to the development of ChIP-seq, native (non-crosslinking) protocols for ChIP (N-ChIP) have been established, which utilize nuclease digestion (often using&#160;micrococcal nuclease or MNase) to fragment the chromatin, as opposed to sonication performed in traditional crosslinking ChIP techniques9. However, one major drawback to both crosslinking ChIP and N-ChIP technologies has been the requirement for high cell numbers due to low DNA yield following the experimental manipulation. Therefore, in more recent years, many efforts have been toward optimizing ChIP technologies for low cell input. These efforts have resulted in the development of many powerful ChIP-based technologies that vary in their applicability and input requirements10,11,12,13,14,15,16,17,18. However, single-cell ChIP-seq based technologies have been lacking, especially for non-histone proteins.
In 2004, an alternative technology was developed to determine protein occupancy on chromatin termed Chromatin Endogenous Cleavage (ChEC) and Chromatin Immunocleavage (ChIC)19. These single-locus techniques utilize a fusion of MNase to either the protein of interest (ChEC) or to protein A (ChIC) for direct cutting of DNA adjacent to the protein of interest. In more recent years, both ChEC and ChIC have been optimized for genome-wide protein profiling on chromatin (ChEC-seq and CUT&#38;RUN, respectively)20,21. While ChEC-seq is a powerful technique for determining factor localization, it requires developing MNase-fusion proteins for each target, whereas ChIC and its genome-wide variation, CUT&#38;RUN, rely on an antibody directed toward the protein of interest (as with ChIP) and recombinant Protein A-MNase, where the Protein A can recognize the IgG constant region of the antibody. As an alternative, a fusion Protein A/Protein G-MNase (pA/G-MNase) has been developed that can recognize a broader range of antibody constant regions22. CUT&#38;RUN has rapidly become a popular alternative to ChIP-seq for determining protein localization on chromatin genome-wide.
Ultra-low input CUT&#38;RUN (uliCUT&#38;RUN), a variation of CUT&#38;RUN that enables the use of low and single-cell inputs, was described in 201923. Here, the methodology for a manual 96-well format single-cell application is described. It is important to note that since the development of uliCUT&#38;RUN, two alternatives for histone profiling, CUT&#38;Tag and iACT-seq have been developed, providing robust and highly parallel profiling of histone proteins24,25. Furthermore, scCUT&#38;Tag has been optimized for profiling multiple factors in a single cell (multiCUT&#38;Tag) and for application to non-histone proteins26. Together, CUT&#38;RUN provides an attractive alternative to low input ChIP-seq where uliCUT&#38;RUN can be performed in any molecular biology lab that has access to a cell sorter and standard equipment.

<table><tbody><tr><td>1.5 mL clear microfuge tubes</td><td>ThermoFisher Scientific</td><td>90410</td><td></td></tr><tr><td>1.5 mL tube magnetic rack</td><td>ThermoFisher Scientific</td><td>12321D</td><td></td></tr><tr><td>1.5 mL tube-compatible cold centrifuge</td><td>Eppendorf</td><td>5404000537</td><td></td></tr><tr><td>10 cm sterile tissue culture plates</td><td>ThermoFisher Scientific</td><td>150464</td><td></td></tr><tr><td>10X T4 DNA Ligase buffer</td><td>New England Biolabs</td><td>B0202S</td><td></td></tr><tr><td>15 mL conical tubes</td><td>VWR</td><td>89039-656</td><td></td></tr><tr><td>1X TE buffer</td><td>ThermoFisher Scientific</td><td>12090015</td><td></td></tr><tr><td>200 &amp;micro;L PCR tubes</td><td>Eppendorf</td><td>951010022</td><td></td></tr><tr><td>2X quick ligase buffer</td><td>New England Biolabs</td><td>M2200</td><td>Ligase Buffer</td></tr><tr><td>5X KAPA HiFi buffer</td><td>Roche</td><td>7958889001</td><td>5X high fidelity PCR buffer</td></tr><tr><td>7-Amino-Actinomycin D (7-AAD)</td><td>Fisher Scientific</td><td>BDB559925</td><td></td></tr><tr><td>96-well magnetic rack</td><td>ThermoFisher Scientific</td><td>12027 or 12331D</td><td></td></tr><tr><td>96-well plate</td><td>VWR</td><td>82006-636</td><td></td></tr><tr><td>AMPure XP beads</td><td>Beckman Coulter</td><td>A63881</td><td>polystyrene-magnetite beads; Due to potential variability between AMPure XP bead lots, it is recommended that your AMPure bead solution be calibrated. See manufacturer&amp;rsquo;s instructions</td></tr><tr><td>Antibody to protein of interest</td><td>varies</td><td></td><td></td></tr><tr><td>ATP</td><td>ThermoFisher Scientific</td><td>R0441</td><td></td></tr><tr><td>BioMag Plus Concanavalin A beads</td><td>Polysciences</td><td>86057-10</td><td>ConA-conjugated paramagnetic microspheres</td></tr><tr><td>BSA</td><td></td><td></td><td></td></tr><tr><td>Calcium Chloride (CaCl2)</td><td>Fisher Scientific</td><td>AAJ62905AP</td><td></td></tr><tr><td>Cell sorter</td><td>BD FACSAria II cell sorter</td><td></td><td>Requires training</td></tr><tr><td>Cell-specific media for cell culture</td><td>Varies</td><td></td><td></td></tr><tr><td>Chloroform</td><td>ThermoFisher Scientific</td><td>C298-500</td><td>Chloroform is a skin irritant and harmful if swallowed; handle in a chemical fume hood using gloves, a lab coat, and goggles</td></tr><tr><td>Computer with 64-bit processer and access to a super computing cluster</td><td></td><td></td><td>For computational analyses of resulting sequencing datasets</td></tr><tr><td>DNA spin columns</td><td>Epoch Life Sciences</td><td>1920-250</td><td></td></tr><tr><td>dNTP set</td><td>New England Biolabs</td><td>N0446S</td><td></td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E3889</td><td></td></tr><tr><td>Electrophoresis equipment</td><td>varies</td><td></td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>22032601</td><td>100% vol/vol ethanol is highly flammable; handle in a chemical fume hood using gloves, a lab coat, and goggles</td></tr><tr><td>Ethylenediaminetetraacetic acid&amp;nbsp; (EDTA)</td><td>Fisher Scientific</td><td>BP2482100</td><td></td></tr><tr><td>FBS</td><td>Sigma Aldrich</td><td>F2442</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-1</td><td></td></tr><tr><td>Glycogen</td><td>VWR</td><td>97063-256</td><td></td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-500</td><td></td></tr><tr><td>Heterologous S. cerevisiae DNA spike-in</td><td>homemade</td><td></td><td>Prepared from crosslinked, MNase-digested, and agarose gel extracted genomic DNA purified of protein/RNA and diluted to 10 ng/mL. We recommend yeast genomic DNA, but other organisms can be used if needed.</td></tr><tr><td>Hydrochloric Acid&amp;nbsp; (HCl)</td><td>Fisher Scientific</td><td>A144-212</td><td>Hydrochloric Acid is very corrosive; handle in a chemical fume hood using gloves, a lab coat, and goggles</td></tr><tr><td>Ice Bucket</td><td>varies</td><td></td><td></td></tr><tr><td>Illumina Sequencing platform (e.g., NextSeq500)</td><td>Illumina</td><td></td><td></td></tr><tr><td>Incubator with temperature and atmosphere control</td><td>ThermoFisher Scientific</td><td>51030284</td><td></td></tr><tr><td>KAPA HotStart HiFi DNA Polymerase with 5X KAPA HiFi buffer</td><td>Roche</td><td>7958889001</td><td>hotstart high fidelity polymerase</td></tr><tr><td>Laminar flow hood</td><td>Bakery Company</td><td>SG404</td><td></td></tr><tr><td>Manganese Chloride (MnCl2)</td><td>Sigma Aldrich</td><td>244589</td><td></td></tr><tr><td>Micropipette set</td><td>Rainin</td><td>30386597</td><td></td></tr><tr><td>Minifuge</td><td>Benchmark Scientific</td><td>C1012</td><td></td></tr><tr><td>NEB Adaptor</td><td>New England Biolabs</td><td>E6612AVIAL</td><td>Adaptor</td></tr><tr><td>NEB Universal primer</td><td>New England Biolabs</td><td>E6611AVIAL</td><td>Universal Primer</td></tr><tr><td>NEBNext Multiplex Oligos for Illumina kit</td><td>New England Biolabs</td><td>E7335S/L, E7500S/L, E7710S/L, E7730S/L</td><td>Indexed Primers</td></tr><tr><td>Negative control antibody</td><td>Antibodies-Online</td><td>ABIN101961</td><td></td></tr><tr><td>Nuclease Free water</td><td>New England Biolabs</td><td>B1500S</td><td></td></tr><tr><td>PCR thermocycler</td><td>Eppendorf</td><td>2231000666</td><td></td></tr><tr><td>Phase lock tubes</td><td>Qiagen</td><td>129046</td><td></td></tr><tr><td>Phenol/Chloroform/Isoamyl Alcohol (PCI)</td><td>ThermoFisher Scientific</td><td>15593049</td><td>Phenol is harmful if swallowed or upon skin contact; handle in a chemical fume hood using gloves, a lab coat, and goggles</td></tr><tr><td>Phsophate buffered saline (PBS)</td><td>ThermoFisher Scientific</td><td>10814010</td><td></td></tr><tr><td>Pipette aid</td><td>Drummond Scientific</td><td># 4-000-100</td><td></td></tr><tr><td>Polyethylene glycol (PEG) 4000</td><td>VWR</td><td>A16151</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma Aldrich</td><td>P3911</td><td></td></tr><tr><td>Potassium Hydroxide (KOH)</td><td>Fisher Scientific</td><td>P250-1</td><td>CAUTION KOH is an eye/skin irritant as a solid and corrosive in solution. Handle in a chemical fume hood using gloves, a lab coat, and goggles</td></tr><tr><td>Protease Inhibitors</td><td>ThermoFisher Scientific</td><td>78430</td><td></td></tr><tr><td>ProteinA/G-MNase</td><td>Epicypher</td><td>15-1016</td><td>pA/G-MNase</td></tr><tr><td>ProteinA-MNase, purified from pK19pA-MN</td><td>Addgene</td><td>86973</td><td></td></tr><tr><td>Proteinase K</td><td>New England Biolabs</td><td>P8107S</td><td></td></tr><tr><td>Qubit 1X dsDNA HS Assay Kit</td><td>ThermoFisher Scientific</td><td>Q33230</td><td></td></tr><tr><td>Qubit Assay tubes</td><td>ThermoFisher Scientific</td><td>Q32856</td><td></td></tr><tr><td>Qubit Fluorometer</td><td>ThermoFisher Scientific</td><td>Q33238</td><td></td></tr><tr><td>Quick Ligase with 2X Quick Ligase buffer</td><td>New England Biolabs</td><td>M2200S</td><td></td></tr><tr><td>RNase A</td><td>New England Biolabs</td><td>T3010</td><td></td></tr><tr><td>Sodium Acetate&amp;nbsp; (NaOAc)</td><td>ThermoFisher Scientific</td><td>BP333-500</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma Aldrich</td><td>S5150-1L</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>ThermoFisher Scientific</td><td>BP166-500</td><td>SDS is poisonous if inhaled; handle with care in well ventilated spaces using gloves, eye protection, and an N95-grade respirator when handling</td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Fisher Scientific</td><td>S318-1</td><td>NaOH is an eye/skin irritant as a solid and corrosive in solution. Handle in a chemical fume hood using gloves, a lab coat, and goggles</td></tr><tr><td>Spermidine</td><td>Sigma Aldrich</td><td>S2626</td><td></td></tr><tr><td>Standard Inverted Light Microscope</td><td>Leica</td><td>11526213</td><td></td></tr><tr><td>Standard lab agarose gel materials</td><td>Varies</td><td></td><td></td></tr><tr><td>Standard lab materials such as serological pipettes and pipette tips</td><td>Varies</td><td></td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>T4 DNA Polymerase</td><td>New England Biolabs</td><td>M0203S</td><td></td></tr><tr><td>T4 PNK</td><td>New England Biolabs</td><td>M0201S</td><td></td></tr><tr><td>Tabletop vortexer</td><td>Fisher Scientific</td><td>2215414</td><td></td></tr><tr><td>Taq DNA Polymerase</td><td>New England Biolabs</td><td>M0273S</td><td></td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td>5384000020</td><td>Alternatively, can use a waterbath</td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-5</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>9002-93-1</td><td>Triton X-100 is hazardous; use a lab coat, gloves, and goggles when handling</td></tr><tr><td>Trypsin</td><td>Fisher Scientific</td><td>MT25052</td><td></td></tr><tr><td>Tube rotator</td><td>VWR</td><td>10136084</td><td></td></tr><tr><td>USER enzyme</td><td>New England Biolabs</td><td>M5505S</td><td></td></tr></tbody></table>

single-cell factor localization on chromatin using ultra-low input cleavage under targets and release using nuclease

Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin Immunoprecipitation (ChIP) has been the gold standard for determining protein localization for over 30 years and is defined by the use of an antibody to pull out the protein of interest from sonicated or enzymatically digested chromatin. More recently, antibody tethering techniques have become popular for assessing protein localization on chromatin due to their increased sensitivity. Cleavage Under Targets &amp; Release Under Nuclease (CUT&amp;RUN) is the genome-wide derivative of Chromatin Immunocleavage (ChIC) and utilizes recombinant Protein A tethered to micrococcal nuclease (pA-MNase) to identify the IgG constant region of the antibody targeting a protein of interest, therefore enabling site-specific cleavage of the DNA flanking the protein of interest. CUT&amp;RUN can be used to profile histone modifications, transcription factors, and other chromatin-binding proteins such as nucleosome remodeling factors. Importantly, CUT&amp;RUN can be used to assess the localization of either euchromatic- or heterochromatic-associated proteins and histone modifications. For these reasons, CUT&amp;RUN is a powerful method for determining the binding profiles of a wide range of proteins. Recently, CUT&amp;RUN has been optimized for transcription factor profiling in low populations of cells and single cells and the optimized protocol has been termed ultra-low input CUT&amp;RUN (uliCUT&amp;RUN). Here, a detailed protocol is presented for single-cell factor profiling using uliCUT&amp;RUN in a manual 96-well format.

Here, a detailed protocol is presented for single-cell protein profiling on chromatin using a 96-well manual format uliCUT&#38;RUN. While results will vary based on the protein being profiled (due to protein abundance and antibody quality), cell type, and other contributing factors, anticipated results for this technique are discussed here. Cell quality (cell appearance and percent of viable cells) and single-cell sorting should be assessed prior to or at the time of live-cell sorting into the NE buffer. An example of ES...

Watch this Scientific Journal Video about Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease at JoVE.com

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

CUT&#38;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&#38;RUN.

Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin ...

single-cell-factor-localization-on-chromatin-using-ultra-low-input

Research

JoVE Journal

Biology

2.6K Views.  University of Pittsburgh. CUT&RUN is een krachtige techniek voor het bepalen van eiwitlokalisatie op chromatine, en hier beschrijven we een eencellige versie van deze techniek in een handmatig 96-well formaat. De meeste eiwitlokalisatietechnieken, zoals ChIP-seq, vereisen een hoge celinvoer en duren ongeveer drie dagen om te voltooien. CUT&RUN is vanwege het hoge signaal en de lage achtergrond geoptimaliseerd voor toepassingen met lage cellen en een cellen en kan in één dag worden voltooid.We stellen ons voor...

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

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Genetics

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Capturing Chromosome Conformation Across Length Scales

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) is used to fix chromatin interactions. Then, chromatin digestion with a restriction enzyme and subsequent religation of fragment ends converts three-dimensional (3D) proximity into unique ligation products. Finally, after reversal of crosslinks, protein removal, and DNA isolation, DNA is sheared and prepared for high-throughput sequencing. The frequency of proximity ligation of pairs of loci is a measure of the frequency of their colocalization in three-dimensional space in a cell population.
A sequenced Hi-C library provides genome-wide information on interaction frequencies between all pairs of loci. The resolution and precision of Hi-C relies on efficient crosslinking that maintains chromatin contacts and frequent and uniform fragmentation of the chromatin. This paper describes an improved in situ Hi-C protocol, Hi-C 3.0, that increases the efficiency of crosslinking by combining two crosslinkers (formaldehyde [FA] and disuccinimidyl glutarate [DSG]), followed by finer digestion using two restriction enzymes (DpnII and DdeI). Hi-C 3.0 is a single protocol for the accurate quantification of genome folding features at smaller scales such as loops and topologically associating domains (TADs), as well as features at larger nucleus-wide scales such as compartments.

Hi-C 3.0 is an improved Hi-C protocol that combines formaldehyde and disuccinimidyl glutarate crosslinkers with a cocktail of DpnII and DdeI restriction enzymes to increase the signal-to-noise ratio and the resolution of chromatin interaction detection.

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) ...

A Comprehensive Approach to Studying the Promoter Interactome in Scarce Cells

An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity with their target gene promoters to control transcription. Although these regulatory elements are easy to identify, their target genes are difficult to predict, since most of them are cell-type specific and may be separated by hundreds of kilobases in the linear genome sequence, skipping over other non-target genes. For several years, Promoter Capture Hi-C (PCHi-C) has been the gold standard for the association of distal regulatory elements to their target genes. However, PCHi-C relies on the availability of millions of cells, prohibiting the study of rare cell populations such as those commonly obtained from primary tissues. To overcome this limitation, low input Capture Hi-C (liCHi-C), a cost-effective and customizable method to identify the repertoire of distal regulatory elements controlling each gene of the genome, has been developed. liCHi-C relies on a similar experimental and computational framework as PCHi-C, but by employing minimal tube changes, modifying&#160;the reagent concentration and volumes, and swapping or eliminating steps, it accounts&#160;for minimal material loss during library construction. Collectively, liCHi-C enables the study of gene regulation and spatiotemporal genome organization in the context of developmental biology and cellular function.

Author Spotlight: An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations  

Gene expression is regulated by interactions of gene promoters with distal regulatory elements. Here, we descirbe how low input Capture Hi-C (liCHi-C) allows the identification of these interactions in rare cell types, which were previously unmeasurable.

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity ...

Mapping Mammalian 3D Genome Interactions with Micro-C-XL

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, known as enhancers, are thought to regulate the activity of distal promoters via interaction in 3D space. Genome-wide chromosome conformation capture (3C)-technologies, such as Hi-C, have transformed our understanding of how genomes are organized in cells. The current understanding of 3D genome organization is limited by the resolution with which the topological organization of chromosomes in 3D space can be resolved. Micro-C-XL measures chromosome folding with resolution at the level of the nucleosome, the basic unit of chromatin, by utilizing micrococcal nuclease (MNase) to fragment genomes during the chromosome conformation capture protocol. This results in an improved signal-to-noise ratio in the measurements, thus facilitating the better detection of insulation sites and chromosome loops compared to other genome-wide 3D technologies. A visually supported, detailed, step-by-step protocol for preparing high-quality Micro-C-XL samples from mammalian cells is presented in this article.

A protocol for mapping the three-dimensional genome organization with nucleosome resolution using the genome-wide chromosome conformation capture method Micro-C-XL is presented here.

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, ...

Locus-Specific Chromatin Isolation from Yeast Cells

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone octamer. Chromatin is defined as the entity of NCPs and numerous other protein complexes, including transcription factors, chromatin remodeling and modifying enzymes. It is still unclear how these protein-DNA interactions are orchestrated at the level of specific genomic loci during different stages of the cell cycle. This is mainly due to the current technical limitations, which make it challenging to obtain precise measurements of such dynamic interactions. Here, we describe an improved method combining site-specific recombination with an efficient single-step affinity purification protocol to isolate a single-copy gene locus of interest in its native chromatin state. The method allows for the robust enrichment of the target locus over genomic chromatin, making this technique an effective strategy for identifying and quantifying protein interactions in an unbiased and systematic manner, for example by mass spectrometry. Further to such compositional analyses, native chromatin purified by this method likely reflects the in vivo situation regarding nucleosome positioning and histone modifications and is, therefore, amenable to further structural and biochemical analyses of chromatin derived from virtually any genomic locus in yeast.

Author Spotlight: Advancing Chromatin Research and Overcoming Limitations with a High-Enrichment Locus-Specific Chromatin Isolation Protocol 

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone ...

Biochemistry

Satellite Cell Isolation from Mouse Limb Muscles and Flow Cytometry Analysis

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion,&#160;the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.

Author Spotlight: Cistrome Analysis in Mouse Muscle Stem Cells 

Presented here is an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells adapted to the study of transcription regulation in muscle fibers by cleavage under targets and release using nuclease (CUT&amp;RUN).

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...

Developmental Biology

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Bioengineering

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

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

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