Figure 1B, 1C, 1E and 1F are reproduced from Bai&#160;et al.10&#160;with permission from the Royal Society of Chemistry. We thank Yi Hao in X. C.&#39;s lab for figure editing. This work is supported by the National Natural Science Foundation of China (No. 91753206 to Q. S. and X. C., No. 31371093 to Q. S., and Nos. 21425204 and 21672013 to X. C.).

&#8203;All animal protocols used in this study were approved by the IACUC (Institutional Animal Care and Use Committee) of Tsinghua University and performed in accordance with guidelines of the IACUC. The laboratory animal facility at Tsinghua University has been accredited by the AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International). For staging of embryos, mid-day of the vaginal plug identified was calculated as embryonic day 0.5 (E0.5).
NOTE: All cells are cultured in the cell incubator under conditions of 37 &#176;C and 5% CO2.
1. Preparation of Mouse Endothelial Culture in Permeable Support Inserts
NOTE: BEND3 cells are maintained according to manufacturer&#39;s instructions.
<ol>
	<li>Prepare BEND3 cell medium (BM) by adding 50 mL of FBS and 5 mL of penicillin-streptomycin into 500 mL of DMEM and mix well.</li>
	<li>Aspirate the medium from the dish and wash the BEND3 cells culture with 1 mL of PBS once. Add 1 mL of 0.25% Trypsin-EDTA into the cells and incubate the cells for 4 min at 37 &#176;C.</li>
	<li>Add 1 mL of BM into the cells to neutralize Trypsin-EDTA and pipette up and down gently to completely dissociate the cells. Transfer the cell suspension into a new 15 mL conical tube and pellet by centrifugation at room temperature (RT) for 5 min at 400 x g.</li>
	<li>Aspirate the supernatant from the tube and resuspend the cells with 9 mL of fresh BM, then add 1 mL of cell suspension into one permeable support insert. Add another 2 mL of fresh BM per well at the bottom chamber of the matrix. Continue to culture the cells for one day.</li>
</ol>
2. Preparation of Mouse Primary Cortical NSPCs Culture
<ol>
	<li>Preparation of culture plate, papain digestion medium, and cortical adherent culture medium (AM)
	<ol>
		<li>Coat 6-well plates with poly-L-lysine (PLL) by adding 1 mL of PLL solution per well into 6-well plates. Then, incubate the plates at RT for 30 min.</li>
		<li>Transfer the PLL solution into a 15 mL conical tube. Wash the plates 3 times with double distilled water. Airdry the plates and put them aside until use.</li>
		<li>Prepare the papain digestion medium by adding 50 U of papain, 50 &#181;L of L-glutamine, and 50 &#181;L of 100 mg/mL acetyl-L-cysteine into 5 mL of DMEM. Mix the medium briefly and warm it to 37 &#176;C for 30 min for enzyme activation.</li>
		<li>Prepare the cortical cell adherent culture medium (AM): add 500 &#181;L of L-glutamine, 500 &#181;L of sodium pyruvate, 500 &#181;L of 100 mg/mL N-acetyl-L-Cysteine, 500 &#181;L of N2, 1 mL of B27, and 5 &#181;L of 100 &#181;g/mL bFGF into 50 mL of DMEM. Mix the medium well and warm it to 37 &#176;C before use.</li>
	</ol>
	</li>
	<li>Preparation of primary cerebral cortical cells and subsequent plating
	<ol>
		<li>Sacrifice an E10.5 timed pregnant mouse by cervical dislocation. 
		NOTE: At E10.5, a majority of cells are proliferating NSPCs in the cerebral cortex, giving rise to large clones of progeny in vitro.</li>
		<li>Sterilize the abdomen by 75% ethanol. Use fine scissors and micro-serrated forceps to open the abdomen by cutting the skin and underlying muscle along the right side of the middle line. Remove the uterus from the abdominal cavity gently with serrated forceps and cut it out from the abdominal cavity with fine scissors.</li>
		<li>Wash the uterus with 40 mL of pre-chilled HBSS in 10 cm Petri dish. Then, transfer the uterus into a new 10 cm Petri dish and wash it again with 40 mL of pre-chilled HBSS.</li>
		<li>Transfer the uterus into a new 10 cm Petri dish with 40 mL of pre-chilled HBSS. Remove the embryos from the uterus and amniotic membrane, then cut the heads of the embryos off from the trunks with Jewelers microforceps.</li>
		<li>Wash the heads with 40 mL of pre-chilled HBSS and transfer the heads to a new 10 cm Petri dish with 40 mL of pre-chilled HBSS. Use Jewelers microforceps to peel away skin and cartilage covering the brains, then cut the cerebral cortices off and collect them in a 15 mL conical tube with pre-chilled HBSS.</li>
		<li>Pellet the cortices by centrifugation for 3 min at 4 &#176;C and 300 x g. Aspirate the supernatant from the tube, then add activated papain digestion&#160;medium and 15 &#181;L of 4 mg/mL DNase I into the tissue pellet.</li>
		<li>Resuspend the tissue pellet briefly by gentle vortexing. Incubate the tissue at 37 &#176;C for 30 min. During this time, loosen the tissue by brief vortexing every 10 min. 
		NOTE: At the end of the digestion, there should be no visible tissue pieces in the tube.</li>
		<li>Pellet the cortical cells by centrifugation for 10 min at 4 &#176;C and 450 x g. Aspirate the supernatant from the tube and wash the cell pellet with pre-chilled DMEM. Repeat this step once. 
		NOTE: During the digestion and washing, take caution not to pipette the tissues and cell pellet roughly to avoid damaging the cells with a strong shearing force.</li>
		<li>Aspirate the supernatant from the tube then add 1.5 mL of pre-chilled HBSS into the tube. Dissociate the cortical cell pellet into single cells with gentle pipetting. Count the cell number with a hemocytometer.</li>
		<li>Add 2 mL of AM and 2 x 104 cortical cells per well into 6-well plates. Incubate the plate at 37 &#176;C and 5% CO2 for 3 h to let the cells&#160;attach to the plate.</li>
	</ol>
	</li>
</ol>
3. Set-up of Neural-endothelial Co-culture and Ac4ManNAz Labeling System
<ol>
	<li>One day after plating BEND3 cells in the inserts, gently aspirate the medium in the bottom chamber first, then the inserts. Wash the enface of the inserts 3 times with pre-warmed DMEM. Wash the outer surface of the inserts by rinsing with pre-warmed DMEM.</li>
	<li>Add 1 mL of pre-warmed AM into one insert, then transfer the inserts into the wells with primary cortical cells. Incubate the co-culture at 37 &#176;C and 5% CO2 for 12 h.</li>
	<li>Dissolve Ac4ManNAz in DMSO to achieve a stock concentration of 200 mM. 12 h after setting up the neural-endothelial co-culture, add 1 &#181;L of Ac4ManNAz stock per bottom chamber and 0.5 &#181;L of stock per insert into the co-culture. Shake the plates immediately and gently to mix the medium well. For the control cells, add equal volume of DMSO.</li>
	<li>Culture the cells for another 5 days at 37 &#176;C and 5% CO2. Prepare the AM with 10x bFGF as refeeding medium (RM). During this time, add 100 &#181;L of RM per insert and 200 &#181;L of RM per bottom chamber to refeed the endothelial and neural cells every other day. During the refeeding, do not supply Ac4ManNAz or DMSO into the culture.</li>
</ol>
4. Immunofluorescent Staining of Sialoglycoproteins in Expanded Primary NSPCs and Differentiated Neurons
<ol>
	<li>Prepare BTTAA-CuSO4 complex 1 30x stock containing 1.5 mM CuSO4 and 9 mM BTTAA in double-distilled water. Prepare freshly biotin-conjugated buffer 1 containing 50 &#181;M biotin-alkyne, 2.5 mM sodium ascorbate, and 1x BTTAA-CuSO4 complex in PBS.</li>
	<li>Remove the inserts from the co-culture plates. Aspirate the culture medium from the bottom wells and wash the neural cells once with pre-warmed PBS.</li>
	<li>Aspirate the PBS from the wells. Add 1 mL of pre-chilled 4% paraformaldehyde PBS solution per well into the cells and fix the cells at RT for 10 min. Then, wash the cells 3 times with pre-chilled PBS.</li>
	<li>Aspirate PBS from the wells and add 1 mL of freshly prepared biotin-conjugated buffer 1 per well into the cells. Incubate the cells at RT for 10 min.</li>
	<li>Aspirate the reaction buffer from the wells. Wash the cells 3 times with PBS. Prepare the staining buffer containing 1% FBS and 1 &#181;g/mL Alexa Fluor 647-streptavidin. Add 1 mL of staining buffer per well into the cells and incubate the cells at RT for 30 min.</li>
	<li>Aspirate the staining buffer from the wells and washed cells 3 times with pre-chilled PBS. Prepare the blocking buffer containing 5% BSA and 0.3% non-ionic detergent-100 in PBS. Add 1 mL of blocking buffer per well into the cells and incubate at RT for 10 min.</li>
	<li>Prepare a primary antibody solution by diluting the anti-nestin and anti-&#946;-tubulin III antibodies together into the blocking buffer at ratios of 1:20 and 1:1,000, respectively. Remove the blocking buffer from the wells and add 1 mL of primary antibody solution per well into the cells. Incubate the cells at 4 &#176;C overnight.</li>
	<li>Remove the primary antibody solution from the wells. Wash the cells 3 times with pre-chilled PBS. Prepare a secondary antibody solution by diluting Alexa Fluor 488 goat anti-mouse IgG1, Alexa Fluor 546 goat anti-mouse IgG2b, and DAPI together into blocking buffer at a dilution of 1:1,000. Aspirate the PBS from the wells and add 1 mL secondary antibody solution per well into cells. Incubate the cells at RT for 2 h.</li>
	<li>Aspirate the antibody solution from the wells and wash the cells 3 times with pre-chilled PBS. Afterwards, the cells are ready for image capture.</li>
</ol>
5. Purification of Sialoglycoproteins from Expanded Primary NSPCs and Differentiated Neurons
<ol>
	<li>Prepare BTTAA-CuSO4 complex 2 15x stock containing 1.5 mM CuSO4 and 3 mM BTTAA in double distilled water. Prepare protein resuspension buffer A containing 4% SDS and 10 mM EDTA in double distilled water; protein resuspension buffer B containing 150 mM NaCl, 50 mM triethanolamine, and 1% polyoxyethylene oleyl ether (e.g., Brij97) in double distilled water with pH 7.4. Before use, mix buffer A:buffer B = 1:8 (vol/vol) to prepare the full protein resuspension buffer. Prepare protein washing buffer 1 containing 2% SDS in PBS; protein washing buffer 2 containing 8 M urea in 250 mM ammonium bicarbonate (ABC); and protein washing buffer 3 containing 2.5 M NaCl in PBS.</li>
	<li>Remove the inserts from the co-culture plates. Aspirate the culture medium from the bottom wells and wash the neural cells once with pre-chilled PBS.</li>
	<li>Aspirate the PBS from the wells and add 200 &#181;L of pre-chilled RIPA buffer per well into the plates. Incubate the plates on ice for 5 min. Collect the protein lysis into 1.5 mL tubes. Pellet the cell debris by centrifugation for 10 min at 4 &#176;C and 12,000 x g.</li>
	<li>Transfer the supernatant into new 1.5 mL tubes. Determine protein concentration with the BCA kit according to the manufacturer&#39;s instructions. Adjust the protein concentration to 1 mg/mL.</li>
	<li>Add 100 &#181;M alkyne-biotin, 2.5 mM sodium ascorbate, and 1x BTTAA-CuSO4 complex 2 to 1 mL of protein lysis and mix the solution well. Incubate the mix at RT for 1 h.</li>
	<li>Transfer the reaction solution into 20 mL of pre-chilled methanol in a 50 mL conical tube. Mix well and incubate at -30 &#176;C overnight to precipitate the proteins.</li>
	<li>Pellet the protein precipitates by centrifugation for 15 min at 4 &#176;C and 4,500 x g. Wash the protein pellet twice with 20 mL of pre-chilled methanol. Aspirate the supernatant from the tube. Resuspend the protein pellet with 4 mL of protein resuspension buffer and transfer the protein resuspension into a new 15 mL conical tube.</li>
	<li>Take 50 &#181;L of streptavidin beads and wash them 3 times with PBS. Add the washed beads into the protein resuspension. Incubate the solution at 4 &#176;C for 3 h on a vertical rotator at a rotation speed of 20 rpm.</li>
	<li>Wash the beads sequentially with 6 types of buffers: protein washing buffer 1, protein washing buffer 2, protein washing buffer 3, 0.5 M ABC, 0.25 M ABC and 0.05 M ABC.</li>
	<li>After washing, resuspend the beads with 20 &#181;L of PBS and transfer the beads into a new 1.5 mL tube. Add 20 &#181;L of 2x protein loading buffer into the beads and treat at 95 &#176;C for 10 min. The protein samples should then be subjected to SDS-PAGE and stained with Coomassie brilliant blue R-250 according to the manufacturer&#39;s instructions. Cut the proteins in gel as indicated by Coomassie brilliant blue R-250 for mass spectrometry analysis.</li>
</ol>

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The authors have nothing to disclosure.

Surface markers are commonly used to label and purify specific cell types in vitro and in vivo17,18. Discovery of surface markers contributes greatly to regenerative medicine and stem cell researches by providing molecular tools to selectively enrich a stem cell population from normal or pathological tissues and culture dishes, offering a purified cell resource for clinical use or study of biological properties. However, progress in developing surface markers for...

Neural stem cells are defined as a multipotent cell population that can self-renew to maintain a stem cell pool and differentiate into neurons and glia. They are the major&#160;cell types in the nervous system and may offer great therapeutic potential in regenerative medicine through cell transplantation into diseased and injured brains1,2. As development proceeds, the neural stem cell population becomes heterogenous3,4, and the proportion of neural stem cells in the brain gradually decreases5. Generally speaking, embryonic neural stem cells and other neural progenitor cells, collectively called neural stem and progenitor cells (NSPCs), are located in the germinal zones, the ventricular zone, and the subventricular zone in mice6. In the embryonic brain, neural stem cells generate neurons directly or indirectly through intermediate progenitor cells (IPCs), and in some species through the outer subventricular zone progenitors (oRGs)7,8. The specific molecular signature, morphology, location in the stem cell niche, and differentiation potential all determine the role of each subtype in brain organogenesis and clinical applications9. However, the currently available cell surface markers cannot unequivocally discriminate and purify different subtypes of NSPCs, limiting the understanding and utilization of these subtypes.
The identification of primary NSPCs surface markers is limited by three major hurdles. The first one is the limited cell number of NSPCs in the tissue, making it difficult&#160;to prepare cell surface protein samples for common mass spectrometry analysis. The second limitation is the difficulty in producing pure cell subtypes for generating subtype-specific membrane protein data. Finally, the third challenge is the low ratio of cell surface proteins in whole cell proteins, which hampers their detection sensitivities by mass spectrometry analysis.
To overcome these problems, we developed a chemoproteomic approach to selectively enrich and identify cell surface proteins in primary NSPCs by metabolically&#160;labeling the sialoglycoproteins10. To generate a sufficient number of NSPCs, we took advantage of an established protocol to expand and maintain primary embryonic NSPCs in undifferentiated states in vitro, by co-culturing NSPCs with mouse brain endothelial cell lines using a permeable support matrix insert (e.g., transwell) system11. In contrast, NPSCs cultured alone without endothelial cells generate differentiated progeny11,12. Thus, protein&#160;samples from these two culture systems can be comparatively analyzed to identify proteins that are differentially expressed in NSPCs and differentiated neurons. As most cell surface proteins are modified by sialic acid13, unnatural sialic acid precursor analog N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz) was used to hijack the intrinsic metabolic pathway so that endogenous, newly synthesized sialoglycans are labeled with azido groups, generating a chemical handle14. Through azido-alkyne-mediated bioorthogonal reactions, which conjugate biotin to sialoglycans, cell surface proteins can be visualized and enriched for proteomic identification through a streptavidin-coupled fluorophore or matrix14.
Here, we perform staining of SDS-PAGE gel analysis of the surface sialoglycoproteome from NSPCs expanded in an endothelial co-culture and differentiating cells in a&#160;non-co-culture system. We also selectively purify surface sialoglycoproteome in the two culture systems for proteomic comparison. Our protocol, compared with&#160;the traditional centrifugation-based cell surface purification protocols15, increases extraction efficacy by reducing the surface protein extraction procedures through specific tag conjugation and affinity purification. Meanwhile, it increases the extraction purity of cell surface proteins based on the premise that sialylation happens mostly at the cell surface proteins. Although endothelial factors cannot completely block differentiation of expanded NSPCs, the comparative study between a co-culture and differentiated culture provides a convenient method to pinpoint stem cell-enriched surface proteins without the need to analyze proteins from NPCs purified by FACS16. We believe this approach can be applied to studies of surface proteins in other systems with the appropriate modifications.

<table border="0" cellpadding="0" cellspacing="0" style="width:880px;" width="879">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">BEND3</td>
			<td style="width:283px;">ATCC</td>
			<td style="width:123px;">CRL-229</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">DMEM</td>
			<td style="width:283px;">Gibco</td>
			<td style="width:123px;">11960044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">L-glutamine</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">25030081</td>
			<td align="right">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sodium pyruvate</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">P5280</td>
			<td align="right">1%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">N2 supplement</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">17502048</td>
			<td>1 to 100</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">N-acetyl-L-cysteine</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">A7250</td>
			<td>1 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">Papain</td>
			<td style="width:283px;">Worthington</td>
			<td style="width:123px;">LS003726</td>
			<td>10 U/mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">B27 supplement</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">17504044</td>
			<td>1 to 50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Poly-L-lysine</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">P4707</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">basic Fibroblast growth factor</td>
			<td style="width:283px;">Gibco</td>
			<td style="width:123px;">PHG0261</td>
			<td>10 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Penicillin-Streptomycin</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">15140122</td>
			<td align="right">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fetal bovine serum</td>
			<td style="width:283px;">Gibco</td>
			<td style="width:123px;">10099141</td>
			<td align="right">10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">HBSS</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">14175095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tripsin-EDTA, 0.25%</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">DPBS</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">14190094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Transwell</td>
			<td style="width:283px;">Corning</td>
			<td align="right" style="width:123px;">3450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Paraformaldehyde</td>
			<td style="width:283px;">Sigma</td>
			<td align="right" style="width:123px;">158127</td>
			<td align="right">4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sucrose</td>
			<td style="width:283px;">Sangon</td>
			<td style="width:123px;">A100335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">DAPI</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">RIPA buffer</td>
			<td style="width:283px;">Thermo Scientific</td>
			<td align="right" style="width:123px;">89900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">SDS-PAGE loading buffer 2X</td>
			<td style="width:283px;">Solarbio</td>
			<td style="width:123px;">P1018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">6-well plate</td>
			<td style="width:283px;">Corning</td>
			<td align="right" style="width:123px;">3335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tris-Glycine protein gel</td>
			<td style="width:283px;">invitrogen</td>
			<td style="width:123px;">xp00100box</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">mouse monoclonal anti-Nestin</td>
			<td style="width:283px;">Developmental Study Hybridoma Bank</td>
			<td style="width:123px;">Rat-401</td>
			<td style="width:172px;">1 to 20</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">mouse monoclonal anti-beta-tubulin III</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">T8860</td>
			<td>1 to 1000</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">Alexa Fluor 488 goat anti-mouse IgG1</td>
			<td style="width:283px;">invitrogen</td>
			<td style="width:123px;">A-21121</td>
			<td>1 to 1000</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">Alexa Fluor 546 goat anti-mouse IgG2b</td>
			<td style="width:283px;">invitrogen</td>
			<td style="width:123px;">A-21143</td>
			<td>1 to 1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Albumin Bovine V</td>
			<td style="width:283px;">Amresco</td>
			<td style="width:123px;">0332</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Triton X-100</td>
			<td style="width:283px;">Amresco</td>
			<td style="width:123px;">0694</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">BCA assay kit</td>
			<td style="width:283px;">Thermo Scientific</td>
			<td align="right" style="width:123px;">23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">dimethyl sulfoxide</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Brij97</td>
			<td style="width:283px;">Aladdin</td>
			<td style="width:123px;">B129088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">CuSO4</td>
			<td style="width:283px;">Sigma</td>
			<td align="right" style="width:123px;">209198</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">alkyne-biotin</td>
			<td style="width:283px;">Click Chemistry Tools</td>
			<td style="width:123px;">TA105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">BTTAA</td>
			<td style="width:283px;">Click Chemistry Tools</td>
			<td align="right" style="width:123px;">1236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ac4ManNAz</td>
			<td style="width:283px;">Click Chemistry Tools</td>
			<td align="right" style="width:123px;">1084</td>
			<td>100 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">9AzSia</td>
			<td style="width:283px;">synthesized in lab</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">sodium ascorbate</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">A4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Methanol</td>
			<td style="width:283px;">Sigma</td>
			<td align="right" style="width:123px;">34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">EDTA</td>
			<td style="width:283px;">Sangon</td>
			<td style="width:123px;">A100322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">NaCl</td>
			<td style="width:283px;">Sangon</td>
			<td style="width:123px;">A100241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">SDS</td>
			<td style="width:283px;">Sangon</td>
			<td style="width:123px;">A100227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Alexa Flour 647-conjugated streptavidin</td>
			<td style="width:283px;">invitrogen</td>
			<td style="width:123px;">S21374</td>
			<td>1 to 1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Triethanolamine</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">V900257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Dynabeads M-280 Streptavidin&#160;</td>
			<td style="width:283px;">invitrogen</td>
			<td align="right" style="width:123px;">60210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">ammonium bicarbonate</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">9830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Coomassie Brilliant Blue R-250</td>
			<td style="width:283px;">Thermo Scientific</td>
			<td align="right" style="width:123px;">20278</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Isoflurane</td>
			<td style="width:283px;">RWD Life Science Co.</td>
			<td style="width:123px;">970-00026-00</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">DNase I</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">DN25</td>
			<td>12 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">urea</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">U5378</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying cell surface markers of primary neural stem and progenitor cells by metabolic labeling of sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

The whole procedure for in vitro expansion and metabolic labeling of primary embryonic NSPCs takes 6 days (Figure 1A). Quality of the BEND3 cell line and freshly isolated primary NSPCs are key to a successful experiment. BEND3 cells are the source of soluble factors that stimulate self-renewal and proliferation of NSPCs. It should be ensured that the BEND3 cells are free of any contamination and divide actively with minimal cell death before co-culturing with...

Watch this Scientific Journal Video about Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan at JoVE.com

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

identifying-cell-surface-markers-primary-neural-stem-progenitor-cells

Research

JoVE Journal

Neuroscience

5.2K Views.  Tongji University and Frontier Science Research Center for Stem Cells of Ministry of Education. Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Video: Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFR&#945;+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis

In mammalian cells, gene transcription is regulated in a cell type specific manner by the interactions of transcriptional factors with genomic DNA. Lineage-specific transcription factors are considered to play essential roles in cell specification and differentiation during development. ChIP coupled with high-throughput DNA sequencing (ChIP-seq) is widely used to analyze genome-wide binding sites of transcription factors (or its associated complex) to genomic DNA. However, a large number of cells are required for one standard ChIP reaction, which makes it difficult to study the limited number of isolated primary cells or rare cell populations. In order to understand the regulatory mechanism of oligodendrocyte lineage-specific transcription factor Olig2 in acutely purified mouse OPCs, a detailed method using ChIP-seq to identify the genome-wide binding sites of Olig2 (or Olig2 complex) is shown. First, the protocol explains how to purify the platelet-derived growth factor receptor alpha (PDGFR&#945;) positive OPCs from mouse brains. Next, Olig2 antibody mediated ChIP and library construction are performed. The last part describes the bioinformatic software and procedures used for Olig2 ChIP-seq analysis. In summary, this paper reports a method to analyze the genome-wide bindings of transcriptional factor Olig2 in acutely purified brain OPCs.

Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFR&amp;#945;+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis

Here we present a protocol which is designed to analyze the genome-wide binding of the oligodendrocyte transcription factor 2 (Olig2) in acutely purified brain oligodendrocyte precursor cells (OPCs) by performing low-cell chromatin immunoprecipitation (ChIP), library preparation, high-throughput sequencing and bioinformatic data analysis.

In mammalian cells, gene transcription is regulated in a cell type specific manner by the interactions of transcriptional factors with genomic DNA. ...