Name: single-cell factor localization on chromatin using ultra-low input cleavage under targets and release using nuclease
Uploaded: 2022-02-01T15:00:00
Description: 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

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-conju...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+has+a+local+effect+on+transcription+and+histone+acetylation.">DNA methylation has a local effect on transcription and histone acetylation.</a> Molecular and Cellular Biology. 22 (19), 6689-6696 (2002).</li><li>Albert, I., 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=Translational+and+rotational+settings+of+H2A.Z+nucleosomes+across+the+Saccharomyces+cerevisiae+genome.">Translational and rotational settings of H2A.Z nucleosomes across the Saccharomyces cerevisiae genome.</a> Nature. 446 (7135), 572-576 (2007).</li><li>Blat, Y., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cohesins+bind+to+preferential+sites+along+yeast+chromosome+III,+with+differential+regulation+along+arms+versus+the+centric+region.">Cohesins bind to preferential sites along yeast chromosome III, with differential regulation along arms versus the centric region.</a> Cell. 98 (2), 249-259 (1999).</li><li>Ren, 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=Genome-wide+location+and+function+of+DNA+binding+proteins.">Genome-wide location and function of DNA binding proteins.</a> Science. 290 (5500), 2306-2309 (2000).</li><li>Rhee, H. 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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. 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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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;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&#8217;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&#160; (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&#160; (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&#160; (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 ...

CUT&RUN is a powerful technique for determining protein localization on chromatin, and here we describe a single-cell version of this technique in a manual 96-well format. Most protein localization techniques, like ChIP-seq, require high cell input and take approximately three days to complete. CUT&RUN, because of the high signal and low background, has been optimized for low-cell and single-cell application, and can be completed in a single day.We envision that this technique can be applied to almost any biological sample and can be especially powerful to determine factor localization in rare and low-abundance samples, such as precious patient samples. Before performing any single-cell experiments, test the antibody and technique on a high number of non-precious cells. The main limitation of this experiment is the dependence on a robust antibody.Demonstrating the procedure is Santana Lardo, an experienced research specialist from my laboratory. After sorting single cells and binding nuclei to beads, place the 96-well plate onto a 96-well magnetic rack and allow the beads to bind for a minimum of five minutes. Then, remove and discard the supernatant.Add 100 microliters of blocking buffer to the nucleus-bound beads, mix with gentle pipetting, and incubate for five minutes at room temperature. Place the plate on a 96-well magnetic rack and allow the supernatant to clear for a minimum of five minutes. Then, remove and discard the supernatant without disturbing the beads.Remove the plate from the magnetic rack and resuspend the beads in 100 microliters of wash buffer per reaction with gentle pipetting. Place the plate back on the 96-well magnetic rack, allow the supernatant to clear, and then remove and discard the supernatant. Resuspend the beads in 25 microliters of wash buffer per reaction with gentle pipetting.Make a primary antibody master mixture by aliquoting 25 microliters of wash buffer per reaction and then add 0.5 microliters of antibody per reaction. Add 25.5 microliters of the antibody wash buffer mix, followed by gentle pipetting to each sample being treated with an antibody targeting the protein of interest. Incubate for one hour at room temperature.Then, again place the samples back on the 96-well magnetic rack. Once the supernatant is clear, remove and discard it without disturbing beads. After removing the plate from the magnetic rack, wash the beads with 100 microliters of wash buffer and resuspend by pipetting.After placing the plate back on a 96-well magnetic rack and discarding supernatant as demonstrated previously, resuspend each sample in 25 microliters of wash buffer. Make a protein A MNase master mixture by adding 25 microliters of wash buffer and an optimized amount of protein A MNase per reaction. Add 25 microliters of protein A MNase master mixture to each sample, including the control samples.Incubate the samples for 30 minutes at room temperature. Place the plate on a 96-well magnetic rack. Allow the supernatant to clear for a minimum of five minutes and then remove and discard the supernatant without disturbing the beads.After removing the plate from the magnetic rack, wash the beads with 100 microliters of wash buffer and resuspend by gentle pipetting. Place the plate on a 96-well magnetic rack and discard the supernatant as demonstrated previously. Then, remove the samples from the magnetic rack and resuspend the beads in 50 microliters of wash buffer by gentle pipetting.Equilibrate the samples to zero degrees Celsius in an ice water mixture for five minutes and then remove the samples from the ice water bath. Add one microliter of 100-millimolar calcium chloride using a multichannel pipette. Mix well three to five times with gentle pipetting using a large-volume multichannel pipette and then return the samples to zero degrees Celsius.Start a 10 minute timer as soon as the plate is back in the ice water bath. Stop the reaction by pipetting 50 microliters of a solution containing twice the concentration of RSTOP+and buffer into each well in the same order as the calcium chloride was added. Incubate the samples for 20 minutes at 37 degrees Celsius.Spin the plate at 16, 000 times G for five minutes at four degrees Celsius. Place the plate on a 96-well magnetic rack. Allow the supernatant to clear for a minimum of five minutes.Then, transfer supernatants to a fresh 96-well plate and discard the beads. For DNA extraction, add one microliter of 10%SDS and 0.83 microliters of 20 milligrams per milliliter proteinase K to each sample and mix the samples by gentle pipetting. Incubate the samples for 10 minutes at 70 degrees Celsius.Return the plate to room temperature. Add 46.6 microliters of five-molar sodium chloride and 90 microliters of 50%PEG4000 and mix by gentle pipetting. Add 33 microliters of polystyrene magnetite beads to each sample and incubate for 10 minutes at room temperature.Place the plate on a magnetic rack and allow the supernatant to clear for around five minutes. Then, carefully discard the supernatant without disturbing the beads. Rinse twice with 150 microliters of 80%ethanol without disturbing the beads.Spin the plate briefly at 1000 times G for 30 seconds. Place the plate back on a 96-well magnetic rack and remove all residual ethanol without disturbing the beads. Air dry the samples for around two to five minutes.Resuspend the beads in 37.5 microliters of 10-millimolar TRIS hydrochloride of pH 8 and incubate for five minutes at room temperature. Place the plate back on a magnetic rack and allow the beads to bind for five minutes. Transfer 36.5 microliters of the supernatant to a fresh thermocycler-compatible 96-well plate and discard the beads.After performing cell quality assessment, cell appearance and percentage were examined, demonstrating that low-quality embryonic stem cells should not be used. Single-cell sorting was performed using Hoechst 33342 stain, and the test cells were counted to assure either zero or one cell is found in each well. Agarose gel analysis revealed a low molecular weight ladder and individual single-cell uliCUT&RUN libraries.Suboptimal and optimal library analysis show a low-molecular weight ladder, suboptimal library due to inefficient Mnase digestion, and successful library with appropriate digestion. Fragment analyzer distribution shows that the expected size of single-cell ranges from 150 to 500 base pairs. However, in large proteins, DNA will have around 270 base pairs.Protein occupancy using single-locus genome browser depicting high cell number or negative control and single-cell CTCF uliCUT&RUN revealed that single-cell largely represented strong peaks, similar to high-cell uliCUT&RUN. Heat maps of single-cell CTCF or negative control revealed a clear pattern of read enrichment directly over established CTCF binding sites in the cells. One-dimensional heat maps show a higher read density over established CTCF binding sites in single-cell libraries relative to surrounding regions and no-antibody controls, demonstrating antibody-specific enrichment at established binding sites.Equilibrating the bead-bound nuclei on the ice water bath and not overheating the sample upon calcium chloride addition is important to prevent over-digestion of the chromatin.

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

Research

JoVE Journal

Biology

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&amp;#945; Promoter Using Gibson Assembly

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...