This work was supported by National Institutes of Health/National Institute of Neurological Disorders &#38; Stroke (NIH/NINDS) Grants R37-NS094804, R01-NS074387, R21-NS091555 to M.G.C.; NIH/NINDS Grants R01-NS076991, R01-NS082311, and R01-NS096756 to P.R.L.; NIH/NINDS R01-EB022563; the Department of Neurosurgery; Leah&#39;s Happy Hearts, and Chad Though Foundation to M.G.C. and P.R.L. RNA Biomedicine Grant F046166 to M.G.C. F.M.M. is supported by an F31 NIH/NINDS-F31NS103500. R.I.Z.-V. is supported by the NIH/ NIGMS grant 5T34GM007821-37.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan.
1. Generation of brain tumor NS and culturing conditions.
<ol>
	<li>Prepare Neural Stem Cell Medium (NSC) with Dulbecco&#39;s Modified Eagle Medium/F12B27 supplement (1x), N2 supplement (1x), and an antimicrobial reagent. Supplement NSC medium on the day of use with human recombinant endothelial growth factor (EGF) and basic-fibroblast growth factor (FGF) at a final concentration of 20 ng/mL each.</li>
	<li>Induce the brain tumor in mouse using the Sleeping Beauty transposon system in which plasmids encoding oncogenes or shRNA targeting tumor suppressors is injected into the lateral ventricles of neonates to generate spontaneous brain tumors9. Select a mouse with a large tumor, which is confirmed with bioluminescence imaging. 
	NOTE: More detailed information about the Sleeping Beauty Transposon System and plasmid constructs used can be found in Calinescu et al.9.
	<ol>
		<li>To image the mice, inject 100 &#181;L of 30 mg/mL luciferin solution intraperitoneally using a 26 gauge needle.</li>
		<li>5 min after injection of luciferin, anesthetize the animal using an anesthesia chamber with oxygen/isoflurane flow set to 2% isoflurane.</li>
		<li>When the animals are anesthetized (usually 2-3 min), place the animals in the bioluminescence chamber with oxygen isoflurane set to 2% isoflurane. If the animals will be under anesthesia for longer than 5 min, use an ophthalmic ointment to prevent dryness while under anesthesia.</li>
		<li>Image the mice using an in vivo optical imaging system with the following parameters: automatic exposure, large binning, and aperture f=1. The parameter to consider it a large tumor is a bioluminescence &#62;106 photons/s/cm2/sr.</li>
	</ol>
	</li>
	<li>Euthanize the mouse with an overdose of isoflurane inhalation anesthetic in a closed chamber, check by toe pinch that the animal is anesthetized, and decapitate the mouse.</li>
	<li>Extract the brain and dissect it as described previously in Calinescu et al.9.</li>
	<li>Place the brain in a 10 cm Petri dish. If the tumor induced expresses a fluorescent protein, use a fluorescent dissecting microscope set to the appropriate fluorescent channel and 0.3X magnification, otherwise identify tumor mass by the differences in tissue color and texture. Use scalpel and forceps to separate the tumor tissue from the normal brain.</li>
	<li>Mince the tumor into small pieces in the Petri dish. Place the dissected tumor in a 1.5 mL tube with 300 &#181;L of the NSC medium.</li>
	<li>Use a disposable plastic pellet pestle that fits to the walls of a conical microcentrifuge tube to gently homogenize the tumor. Add 1 mL of cell detachment medium and incubate the sample for 5 min at 37 &#176;C. 
	NOTE: The use of a pestle may kill some cells. If the reader wants to maximize the cell viability, the use of a pestle may be omitted.</li>
	<li>Pass the cell suspension from step 1.7 through a 70 &#181;m cell strainer and wash the strainer with 25 mL of NSC medium.</li>
	<li>Centrifuge the suspension at 300 x g for 5 min at room temperature, decant the supernatant and re-suspend the pellet into 6 mL of NSC medium.</li>
	<li>Plate the tumor cell suspension from step 1.9 into a T-25 culture flask and culture it at 37 &#176;C in a tissue culture incubator with an atmosphere of 95% air and 5% CO2. Usually after 3 days, there will be a mixture of some dead cells, adhered cells and some cells forming NS which float in the medium.</li>
	<li>Transfer the cell suspension into a 15 mL conical tube, collect only the cells that sink to the bottom using a micropipette and re-plate them onto a T-75 tissue culture flask.</li>
	<li>Maintain the cells at relatively high density (1-3 X 106 cells in a T-25). Passage the cells when they are confluent. 
	NOTE: Confluency is determined by the spheres size. Black centers of large spheres mean that there are dead cells in the center and indicate that the cells should be passaged immediately.</li>
	<li>Add the growth factors (EGF and basic-FGF; 20 ng/mL each) every three days. Change the medium when the cells are not confluent and the medium turns light orange.</li>
	<li>To change the medium, collect the old medium and centrifuge it at 300 x g at room temperature for 5 min and re-suspend the cell pellet in NSC medium supplemented with EGF and FGF at the concentration specified in step 1.1. 
	NOTE: If the spheres are at mid-to high confluency, but not ready to be passaged (sphere size is small with a diameter &#60; 100 &#181;m), then the pellet can be split into two flasks.</li>
	<li>To passage the cells, centrifuge the cells at 300 x g for 5 min at room temperature, decant the supernatant and incubate the cells in cell detachment medium for 5 min at 37 &#176;C.
	<ol>
		<li>Add 5 mL of balanced salt solution to dilute detachment medium and centrifuge it at 300 x g for 5 min at room temperature.</li>
		<li>Decant the supernatant, resuspend the cell pellet in 18 mL of NSC medium and divide the suspension equally into three T-25 flasks.</li>
	</ol>
	</li>
	<li>To freeze the aliquots of cells, dissociate the cells as in step 1.15, and freeze the cells in 1 mL aliquots of 90% fetal bovine serum with 10% dimethyl sulfoxide. Slowly cool down the cells by placing the cells on ice for 10 min, then keeping the cells -20 &#176;C for 30 min, and -80 &#176;C for 1 day. The next day, transfer the cells to a liquid nitrogen storage container.</li>
</ol>
2. Native ChIP
<ol>
	<li>Chromatin preparation
	<ol>
		<li>After making stock of derived NS, culture NS in a T-75 in 15 mL of NSC medium at 37 &#176;C in a tissue culture incubator with an atmosphere of 95% air and 5% CO2 until NS become confluent. One confluent T-75 plate will yield 3-5 X 106 cells.</li>
		<li>When the cells become confluent, centrifuge NS at 300 x g for 5 min, decant the supernatant and add 1 mL of cell dissociation medium. Incubate the cells at 37 &#176;C for 5 min. Add 5 mL of medium and count the cells using a hemocytometer10. 
		NOTE: 1 x 106 cells are needed per immunoprecipitation (IP) reaction. Note that a pre-immune serum or IgG control must also be included11.</li>
		<li>Centrifuge the NS in one 1.5 mL microcentrifuge tube at 300 x g for 5 min, decant the supernatant and re-suspend NS pellet with 1 mL of balanced salt solution and transfer the suspension to low protein binding 1.5 mL microcentrifuge tubes.</li>
		<li>Centrifuge NS at 300 x g for 5 min, decant the supernatant and re-suspend the cell pellet in 95 &#181;L of digestion buffer (50 mM Tris-HCl, pH 8.0, 1 mM CaCl2, 0.2% polyethylene glycol octylphenyl ether) per 1 x 106 cells supplemented with protease inhibitor cocktail at a high concentration (1:100). Immediately pipet the cells up and down to prevent clumping and avoid creating any bubbles.</li>
		<li>Resuspend MNase in 50% glycerol to make 1 mg/mL solution (1.15 units/&#181;L, stored at -20 &#176;C) that will be referred to as &#34;1x&#34; stock. Make a &#34;0.1x&#34; stock by doing a second 1:9 dilution with 50% glycerol (e.g., 900 &#181;L of &#34;1x&#34; MNase and 100 &#181;L of 50% glycerol) and store it at -20 &#176;C.</li>
		<li>Mix 5 &#181;L of &#34;0.1x&#34; MNase and 145 &#181;L of digestion buffer. Keep the enzyme on ice all the times. Add 5 &#181;L of diluted MNase in digestion buffer per 1 x 106 cells. Flick the tube to mix and place the tube in 37 &#176;C block for exactly 12 min. Process the samples one at a time to ensure an accurate incubation time of 12 min.</li>
		<li>Add 10 &#181;L of 10x MNase Stop Buffer (110 mM Tris-HCl, pH 8.0, 55 mM EDTA) per 1 x 106 cells. Samples must be kept on ice from this point onwards.</li>
		<li>Add 110 &#181;L &#34;2x RIPA buffer&#34; (280 mM NaCl, 1.8% polyethylene glycol octylphenyl ether, 0.2% sodium dodecyl sulfate (SDS), 0.2% Na-deoxycholate, 5 mM ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA); stored at 4 &#176;C) supplemented with protease inhibitor cocktail at high concentration (1:100) per 1 x 106 cells. Flick the tube to mix.</li>
		<li>Centrifuge the tubes for 15 min in a microfuge at 4 &#176;C and 1700 x g. Transfer the supernatant to a new regular 1.5 mL microcentrifuge tubes (low protein binding microcentrifuge tubes are no longer necessary) and store them on ice. Discard the pellet.</li>
	</ol>
	</li>
	<li>Immunoprecipitation (IP)
	<ol>
		<li>Mix Protein A and Protein G magnetic beads in a 1:1 ratio. For every IP, prepare 25 &#181;L of beads.</li>
		<li>Wash the beads by adding 1 mL of RIPA buffer (10 mM Tris pH 8.0, 1 mM EDTA, 140 mM NaCl, 1% polyethylene glycol octylphenyl ether, 0.1% SDS, 0.1% Na-deoxycholate; stored at 4 &#176;C) supplemented with protease inhibitors at low concentration (1:1000).</li>
		<li>Use a magnet to allow beads to separate from the washing solution and decant the washing solution (approximately 1 min). Perform the wash step twice.</li>
		<li>Re-suspend the beads to the original volume using RIPA buffer supplemented with protease inhibitors (1:1000).</li>
		<li>If necessary, dilute the chromatin using RIPA buffer supplemented with protease inhibitors (1:1000) so that each IP has a volume of 100-200 &#181;L. Reserve 10% of the volume used per IP of chromatin for the input. 
		NOTE: For example, if 200 &#181;L are used per IP, 20 &#181;L of diluted chromatin should be reserved for the input. For a total of four IPs, total chromatin volume should be diluted to 820 &#181;L.</li>
		<li>Prepare the input. Add 100 &#181;L of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) supplemented with proteinase K (0.5 mg/mL) to the input and incubate the input at 55 &#176;C for 1 h. 
		NOTE: Steps 2.2.6-2.2.7 can be performed along with steps 2.3.4 and 2.4.1.</li>
		<li>Purify the input using a PCR purification kit following the manufacturer&#39;s instructions and elute in 50 &#181;L of elution buffer provided in kit.</li>
		<li>Measure the concentration of the input using a microvolume spectrophotometer and record the results in a notebook. Blank with elution buffer from kit. 
		NOTE: In mouse neurospheres, we used approximately 6 &#181;g of DNA for each IP.</li>
		<li>Run input on a 1% gel or load 1 &#181;L on a DNA Bioanalyzer using standard settings. Store the remainder for use as input in downstream applications.</li>
		<li>Add 10 &#181;L of washed protein A &#38; G magnetic beads for every IP and incubate IP for 1 h at 4 &#176;C. 
		NOTE: For example, add 30 &#181;L of protein A &#38; G magnetic beads for three IPs.</li>
		<li>Place the samples on a magnet and allow beads to separate. Divide chromatin by transferring the previously determined amount into a new 1.5 mL tube.</li>
		<li>Add the antibody and wrap the caps with plastic paraffin film to avoid evaporation. Incubate the samples overnight at 4 &#176;C with rotation at 20 rpm. 
		NOTE: Concentration should be determined empirically following manufacturer recommendations.</li>
		<li>The next day, spin the tubes using a mini centrifuge at room temperature, 2000 x g, for 10 s. Then add 10 &#181;L of beads to each IP and incubate IP for 3 h at 4 &#176;C with rotation at 20 rpm.</li>
	</ol>
	</li>
	<li>IP Washes and Protein Digestion
	<ol>
		<li>Add 150 &#181;L of RIPA buffer supplemented with protease inhibitors at low concentration (1:1000) to each IP and incubate IP at 4 &#176;C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow the magnetic beads to separate. Use a pipet to remove the supernatant. Repeat this tep five washes.</li>
		<li>Add 150 &#181;L of LiCl buffer (250 mM LiCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.5% NP-40, 0.5 % Na-deoxycholate; store at 4 &#176;C) supplemented with protease inhibitors at low concentration (1:1000) to each IP and incubate IP at 4 &#176;C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow magnetic beads to separate. Use a pipet to remove supernatant.</li>
		<li>Add 150 &#181;L of cold TE (no protease inhibitors) and incubate the sample at 4 &#176;C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow magnetic beads to separate. Use a pipet to remove the supernatant.</li>
		<li>After the final washing step, re-suspend the sample in 100 &#181;L of TE buffer supplemented with proteinase K (0.5 mg/mL) and incubate the sample at 55 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>DNA Purification and ChIP Quantitative Real-Time PCR (qPCR)
	<ol>
		<li>Purify immunoprecipitated DNA using a PCR purification kit. After addition of DNA binding buffer from the kit to each sample, place the tube on a magnet and wait until magnetic beads aggregate, then transfer the supernatant to the cleanup column. Follow the manufacturer&#39;s instructions and elute in 50 &#181;L of elution buffer provided in kit.</li>
		<li>To check if the IP is successful, perform a qPCR. Ideally, primers should be designed for positive and negative control regions. 
		NOTE: For example, for an IP performed with H3K4me3 and H3K27me3, the following samples should be run on qPCR: H3K4me3, H3K27me3, and IgG ChIP DNA, input DNA, and a no template control (NTC) using primers for regions to be enriched with H3K4me3 (positive control) and regions to be enriched with H3K4me3 (negative control); likewise, for H3K27me3.</li>
		<li>Follow standard protocols to perform qPCR12. Briefly, perform a qPCR using SYBR green master mix, 0.5 &#181;L of 10 &#181;M working concentration of each forward and reverse primer, and 2 &#181;L of ChIP or input DNA. Read plates using a Real Time PCR system. Primer sequences are provided in Table 2. The Gapdh primer sequences from Hwang et al. were used13.</li>
		<li>Analyze ChIP data relative to the input, i.e., percent input method (% IP)14. Calculate the percent input using the following formulas: 
		Adjusted input = mean ct(input)-log2(DF) 
		where DF is the dilution factor of the starting input and 
		DF = total volume of IP/ volume of input 
		% IP = 100&#215;2^(Adjusted input - Ct (IP)) 
		where Ct is the threshold factor.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Schwartzentruber, J., 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=Driver+mutations+in+histone+H3.3+and+chromatin+remodelling+genes+in+paediatric+glioblastoma.">Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma.</a> Nature. 482 (7384), 226-231 (2012).</li><li>Bjerke, L., 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=Histone+H3.3.+mutations+drive+pediatric+glioblastoma+through+upregulation+of+MYCN.">Histone H3.3. mutations drive pediatric glioblastoma through upregulation of MYCN.</a> Cancer Discov. 3 (5), 512-519 (2013).</li><li>Bender, 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=Reduced+H3K27me3+and+DNA+hypomethylation+are+major+drivers+of+gene+expression+in+K27M+mutant+pediatric+high-grade+gliomas.">Reduced H3K27me3 and DNA hypomethylation are major drivers of gene expression in K27M mutant pediatric high-grade gliomas.</a> Cancer Cell. 24 (5), 660-672 (2013).</li><li>Maleszewska, M., Kaminska, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+glioblastoma+an+epigenetic+malignancy?.">Is glioblastoma an epigenetic malignancy?.</a> Cancers (Basel). 5 (3), 1120-1139 (2013).</li><li>Gilfillan, G. 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=Limitations+and+possibilities+of+low+cell+number+ChIP-seq.">Limitations and possibilities of low cell number ChIP-seq.</a> BMC Genomics. 13, 645 (2012).</li><li>Thorne, A. W., Myers, F. A., Hebbes, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+chromatin+immunoprecipitation.">Native chromatin immunoprecipitation.</a> Methods Mol Biol. 287, 21-44 (2004).</li><li>Turner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mapping Protein/DNA Interactions by Cross-Linking. , (2001).</li><li>Tseng, Z., Wu, T., Liu, Y., Zhong, M., Xiao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+native+chromatin+immunoprecipitation+to+interrogate+histone+variant+protein+deposition+in+embryonic+stem+cells.">Using native chromatin immunoprecipitation to interrogate histone variant protein deposition in embryonic stem cells.</a> Methods Mol Biol. 1176, 11-22 (2014).</li><li>Calinescu, A. A., 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=Transposon+mediumted+integration+of+plasmid+DNA+into+the+subventricular+zone+of+neonatal+mice+to+generate+novel+models+of+glioblastoma.">Transposon mediumted integration of plasmid DNA into the subventricular zone of neonatal mice to generate novel models of glioblastoma.</a> J Vis Exp. (96), (2015).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+cell+number+by+hemocytometry+counting.">Estimation of cell number by hemocytometry counting.</a> CSH Protoc. 2006 (1), (2006).</li><li>Landt, S. G., 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=ChIP-seq+guidelines+and+practices+of+the+ENCODE+and+modENCODE+consortia.">ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia.</a> Genome Res. 22 (9), 1813-1831 (2012).</li><li>Kubista, M., 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=The+real-time+polymerase+chain+reaction.">The real-time polymerase chain reaction.</a> Mol Aspects Med. 27 (2-3), 95-125 (2006).</li><li>Hwang, W. W., 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=Distinct+and+separable+roles+for+EZH2+in+neurogenic+astroglia.">Distinct and separable roles for EZH2 in neurogenic astroglia.</a> Elife. 3, e02439 (2014).</li><li>Haring, M., 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=Chromatin+immunoprecipitation:+optimization,+quantitative+analysis+and+data+normalization.">Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization.</a> Plant Methods. 3, 11 (2007).</li></ol>

The authors have nothing to disclose.

The protocol presented here will enable the user to perform native ChIP on NS derived from genetically engineered brain tumors. In contrast to cross-linking ChIP, this protocol is limited for the study of proteins that associate tightly with DNA6. The number of cells used can be modified as necessary and the protocol can be scaled up. We used 1 X 106 cells per IP, however, native ChIP may also be performed with as little as 4 x 104 cells5. In this prot...

Epigenetic events are frequent in gliomas and likely play an important role in tumor pathogenesis. Indeed, in pediatric high-grade glioma, mutations in genes encoding histone variants H3.3 and H3.1 occur frequently1. The mutations affect histone modifications and have major epigenetic consequences2,3. In the adolescent to adult spectrum, recurrent mutations in isocitrate dehydrogenase gene 1/2 (IDH1/2), a mutation that inhibits &#945;-KG dependent histone and DNA de-methylasaes, and genetic alterations in other chromatin regulators such as ATRX and DAXX occur4. Therefore, it is of critical importance to study how mutations that affect epigenetic regulators alter chromatin structure and regulatory histone modifications, which, in turn, have a dramatic impact of the tumor cells&#39; transcriptome.
Chromatin immunoprecipitation (ChIP) is a powerful tool used to evaluate the impact of epigenetic modifications in the genome5,6,7. In native ChIP, chromatin is digested with micrococcal nuclease (MNase), immunoprecipitated using an antibody raised against the protein of interest, and then DNA is purified from the immunoprecipitated chromatin complex6. Cells are not fixed during the procedure so this technique is only applicable for the study of proteins that interact tightly with DNA6. The absence of cross-linking aids antibody specificity since antibodies are usually raised against unfixed peptides or proteins7. In addition, since there is no cross-linking step, this reduces the chances of fixing transient protein-DNA interactions that are non-specific and not regulatory7,8. ChIP can be used to identify the enrichment of histone modifications in a specific genomic region. Here, we detail a protocol for performing native ChIP in neurospheres (NS) generated from genetically engineered mouse models of glioma.

<table>
	<tbody>
		<tr>
			<td>Agilent 2100 Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2946-90004</td>
			<td>bioanalyzer</td>
		</tr>
		<tr>
			<td>AccuSpin Micro 17R, refrigerated</td>
			<td>Fisher Scientific</td>
			<td>13-100-676</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6</td>
			<td>Taconic</td>
			<td>B6-f</td>
			<td>C57BL/6 mouse</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Aldrich</td>
			<td>22350-6</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>D-Luciferin, Potassium Salt</td>
			<td>Goldbio</td>
			<td>LuckK-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>DiaMag1.5 magnetic rack</td>
			<td>Diagenode</td>
			<td>B04000003</td>
			<td>for magnetic bead washes</td>
		</tr>
		<tr>
			<td>DiaMag Rotator EU</td>
			<td>Diagenode</td>
			<td>B05000001</td>
			<td>rotator</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>11330-057</td>
			<td>NSC component</td>
		</tr>
		<tr>
			<td>Dynabeads Protein A</td>
			<td>Thermo Fisher Scientific</td>
			<td>10001D</td>
			<td>protein A magnetic beads</td>
		</tr>
		<tr>
			<td>Dynabeads Protein G</td>
			<td>Thermo Fisher Scientific</td>
			<td>10003D</td>
			<td>protein G magnetic beads</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>PeproTech</td>
			<td>AF-100-15</td>
			<td>prepare 20 &#956;g/mL stock in 0.1% BSA and aliquot.</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>E-4884</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid) (EGTA)</td>
			<td>Sigma</td>
			<td>E-4378</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Fast SYBR Green Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4385612</td>
			<td>qPCR reagent</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td>for freezing cells</td>
		</tr>
		<tr>
			<td>FGF</td>
			<td>PeproTech</td>
			<td>100-18b</td>
			<td>Prepare 20 &#956;g/mL stock in 0.1% BSA and aliquot.</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11008-13</td>
			<td>for dissection of tumor</td>
		</tr>
		<tr>
			<td>Glycerol, MB Grade</td>
			<td>EMD- Millipore</td>
			<td>356352</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K4me3</td>
			<td>Abcam</td>
			<td>Ab8580</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K27me3</td>
			<td>Millipore</td>
			<td>07-449</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175-103</td>
			<td>balanced salt solution</td>
		</tr>
		<tr>
			<td>Fluriso</td>
			<td>VETone</td>
			<td>501017</td>
			<td>inhalation anesthetic</td>
		</tr>
		<tr>
			<td>Ivis Spectrum</td>
			<td>Perkin-Elmer</td>
			<td>124262</td>
			<td>in vivo optical imaging system</td>
		</tr>
		<tr>
			<td>Hyqtase</td>
			<td>HyClon</td>
			<td>SV3003001</td>
			<td>cell detachment media</td>
		</tr>
		<tr>
			<td>Lithium Chloride</td>
			<td>Sigma</td>
			<td>L8895</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Low binding microtubes</td>
			<td>Corning Costar</td>
			<td>CLS3207</td>
			<td>low protein binding microcentrifuge tube</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Fisher</td>
			<td>21-402-903</td>
			<td>regular microcentrifuge tube</td>
		</tr>
		<tr>
			<td>Micrococcal Nuclease</td>
			<td>Thermo Fisher Scientific, Affymetrix</td>
			<td>70196Y</td>
			<td>each batch may differ; purchase sufficient amount for experiments and aliquot.</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>NSC component</td>
		</tr>
		<tr>
			<td>Normal Rabbit IgG</td>
			<td>Millipore</td>
			<td>12-372</td>
			<td></td>
		</tr>
		<tr>
			<td>Normocin</td>
			<td>Invivogen</td>
			<td>NOL-36-063</td>
			<td>anti-microbial agent, use at 0.1 mg/mL.</td>
		</tr>
		<tr>
			<td>NP-40 (Igepal CA-630)</td>
			<td>Sigma</td>
			<td>18896-50ML</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Kimble Kontes Pellet Pestle</td>
			<td>Fisher Scientific</td>
			<td>K749515-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8340</td>
			<td>aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Protinase K</td>
			<td>Sigma-Aldrich</td>
			<td>P2308</td>
			<td>make 10 mg/mL stock in water; aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>DNA purification kit</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10007-16</td>
			<td>for dissection of tumor</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>VWR</td>
			<td>0241-5KG</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Sodium Deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D670-25G</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl sulfate (SDS)</td>
			<td>Sigma</td>
			<td>L-4390</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Thermo Fisher Scientific</td>
			<td>Bp152-1</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP 151-500</td>
			<td>polyethylene glycol octylphenyl ether</td>
		</tr>
		<tr>
			<td>Standard Mini Centrifuge</td>
			<td>Fisherbrand</td>
			<td>12-006-901</td>
			<td>standard mini centrifuge</td>
		</tr>
		<tr>
			<td>SZX16 microscope</td>
			<td>Olympus</td>
			<td>SZX16</td>
			<td>flourescent dissecting microscope</td>
		</tr>
		<tr>
			<td>ViiA 7 Real-Time PCR System with Fast 96-Well Block</td>
			<td>Applied Biosystems</td>
			<td>4453535</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop One</td>
			<td>Thermo-Fisher Scientific</td>
			<td>ND-ONEC-W</td>
			<td></td>
		</tr>
	</tbody>
</table>

native chromatin immunoprecipitation using murine brain tumor neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

A schematic representation of tumor NS generated from a brain tumor where brain tumor cells are Katushka positive is presented in Figure 1. Figure 2 is a schematic representation of the entire ChIP technique. Figure 3 shows the representative results of chromatin from brain tumor NS digested with MNase for 12 min, yielding a majority of mono, di-, and tri- nucleosomes. Following ChIP, a qPCR may be p...

Watch this Scientific Journal Video about Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres at JoVE.com

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

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

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

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7.6K Views.  University of Michigan Medical School. Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

Video: Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

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

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of associated loci. In addition, the presence of specific modifications has been used to determine the position and identity non-coding functional elements such as enhancers. In recent years, chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) has become a powerful tool in determining the genome-wide profiles of individual histone modifications. However, it has become increasingly clear that the combinatorial patterns of chromatin modifications, referred to as Chromatin States, determine the identity and nature of the associated genomic locus. Therefore, workflows consisting of robust high-throughput (HT) methodologies for profiling a number of histone modification marks, as well as computational analyses pipelines capable of handling myriads of ChIP-Seq profiling datasets, are needed for comprehensive determination of epigenomic states in large number of samples. The HT-ChIP-Seq workflow presented here consists of two modules: 1) an experimental protocol for profiling several histone modifications from small amounts of tumor samples and cell lines in a 96-well format; and 2) a computational data analysis pipeline that combines existing tools to compute both individual mark occupancy and combinatorial chromatin state patterns. Together, these two modules facilitate easy processing of hundreds of ChIP-Seq samples in a fast and efficient manner. The workflow presented here is used to derive chromatin state patterns from 6 histone mark profiles in melanoma tumors and cell lines. Overall, we present a comprehensive ChIP-seq workflow that can be applied to dozens of human tumor samples and cancer cell lines to determine epigenomic aberrations in various malignancies.

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

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of brain tumors, the molecular selectivity of BBB also shields the neoplastic cells by blocking the delivery of peripherally administered chemotherapies. The development of novel drugs (including nanoparticles) targeting malignant brain tumors ideally requires the use of preclinical animal models to study the drug&#8217;s transcytosis and antitumor efficacy. In order to comply with the 3R principle (refine, reduce, and replace) to reduce the number of laboratory animals in experimental setup and perform the high-throughput screening of a large library of antitumor agents, we developed a reproducible in vitro human and murine mimic of the blood-brain tumor-barrier (BBTB) using three-layered cultures of endothelial cells, astrocytes, and patient-derived glioblastoma spheres. For higher scalability and reproducibility, commercial cell lines or immortalized cells have been used in tailored conditions to allow the formation of a barrier resembling the actual BBB. Here we describe a protocol to obtain a BBTB mimic by culturing endothelial cells in contact with astrocytes at specific cell densities on inserts. This BBTB mimic can be used, for instance, for the quantification and confocal imaging of the nanoparticle passage through the endothelial and astrocytic barriers, in addition to the evaluation of the tumor cell targeting within the same assay. Moreover, we show that the obtained data can be used to predict the behavior of nanoparticles in preclinical animal models. In a broader perspective, this in vitro model could be adapted to other neurodegenerative diseases for the determination of the passage of new therapeutic molecules through the BBB and/or be supplemented with brain organoids to directly evaluate the efficacy of drugs.

Drug targeting to central nervous system tumors is a major challenge. Here we describe a protocol to produce an in vitro mimic of the blood-brain tumor-barrier using murine and/or human cells and discuss their relevance for the predictability of central nervous system tumor targeting in vivo.

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of ...

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused on bulk approaches that led to the molecular classification of brain tumors. Over the last five years, single cell sequencing approaches have highlighted several important features of gliomas. The majority of these studies have utilized fresh brain tumor specimens to isolate single cells using flow cytometry or antibody-based separation methods. Moving forward, the use of fresh-frozen tissue samples from biobanks will provide greater flexibility to single cell applications. Furthermore, as the single-cell field advances, the next challenge will be to generate multi-omics data from either a single cell or the same sample preparation to better unravel tumor complexity. Therefore, simple and flexible protocols that allow data generation for various methods such as single-nucleus RNA sequencing (snRNA-seq) and single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq) will be important for the field.
Recent advances in the single cell field coupled with accessible microfluidic instruments such as the 10x genomics platform have facilitated single cell applications in research laboratories. To study brain tumor heterogeneity, we developed an enhanced protocol for the isolation of single nuclei from fresh frozen gliomas. This protocol merges existing single cell protocols and combines a homogenization step followed by filtration and buffer mediated gradient centrifugation. The resulting samples are pure single nuclei suspensions that can be used to generate single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.

Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies.

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...