Name: Author Spotlight: Integrating Organoid Models with Single-Cell and Spatial Transcriptomics Technologies
Uploaded: 2024-03-29T13:30:00
Description: Watch this Scientific Journal Video about Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids at JoVE.com

We thank Valeria Fernandez-Vallone for the original instructions for the Miltenyi Neural Dissociation kit. We also thank the Genomics Technology Platform of the Max Delbrueck Centrum for providing the recipe for the NP40 lysis buffer and valuable advice setting up this protocol. We also thank Margareta Herzog and Alexandra Tschernycheff for the lab organizational support.

The described protocol is performed in a biosafety level 1 laboratory of the Max Delbr&#252;ck Center for Molecular Medicine (approval number: 138/08), in accordance with the requirements and in compliance with EU and national rules on ethics in research.
1. Derivation of forebrain organoids from induced pluripotent stem cells (iPSCs)
NOTE: This protocol was tested for several different iPSC lines cultured in a variety of stem cell media from differen...

Single Cell and Nuclei Extraction from Human Brain Organoids

<ol>
	<li>Finkbeiner, S. R., 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=Stem+cell-derived+human+intestinal+organoids+as+an+infection+model+for+Rotaviruses.">Stem cell-derived human intestinal organoids as an infection model for Rotaviruses.</a> mBio. 3 (4), e00159-e00212 (2012).</li><li>Freedman, B. 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=Modelling+kidney+disease+with+CRISPR-mutant+kidney+organoids+derived+from+human+pluripotent+epiblast+spheroids.">Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids.</a> Nat Commun. 6, 8715 (2015).</li><li>Guan, Y., 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=Human+hepatic+organoids+for+the+analysis+of+human+genetic+diseases.">Human hepatic organoids for the analysis of human genetic diseases.</a> JCI Insight. 2 (17), e94954 (2017).</li><li>Lancaster, M. 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=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 501 (7467), 373-379 (2013).</li><li>Dang, 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=Zika+virus+depletes+neural+progenitors+in+human+cerebral+organoids+through+activation+of+the+innate+immune+receptor+TLR3.">Zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor TLR3.</a> Cell Stem Cell. 19 (2), 258-265 (2016).</li><li>Inak, 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=Defective+metabolic+programming+impairs+early+neuronal+morphogenesis+in+neural+cultures+and+an+organoid+model+of+Leigh+syndrome.">Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome.</a> Nat Commun. 12 (1), 1929 (2021).</li><li>Karlsson, 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=A+single-cell+type+transcriptomics+map+of+human+tissues.">A single-cell type transcriptomics map of human tissues.</a> Sci Adv. 7 (31), eabh2169 (2021).</li><li>Piwecka, M., Rajewsky, N., Rybak-Wolf, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+and+spatial+transcriptomics:+deciphering+brain+complexity+in+health+and+disease.">Single-cell and spatial transcriptomics: deciphering brain complexity in health and disease.</a> Nat Rev Neurol. 19 (6), 346-362 (2023).</li><li>Lim, B., Lin, Y., Navin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+cancer+research+and+medicine+with+single-cell+genomics.">Advancing cancer research and medicine with single-cell genomics.</a> Cancer Cell. 37 (4), 456-470 (2020).</li><li>Camp, J. 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=Human+cerebral+organoids+recapitulate+gene+expression+programs+of+fetal+neocortex+development.">Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.</a> Proc Natl Acad Sci U S A. 112 (51), 15672-15677 (2015).</li><li>Fiorenzano, 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=Single-cell+transcriptomics+captures+features+of+human+midbrain+development+and+dopamine+neuron+diversity+in+brain+organoids.">Single-cell transcriptomics captures features of human midbrain development and dopamine neuron diversity in brain organoids.</a> Nat Commun. 13 (1), 3312 (2022).</li><li>Kanton, 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=Organoid+single-cell+genomic+atlas+uncovers+human-specific+features+of+brain+development.">Organoid single-cell genomic atlas uncovers human-specific features of brain development.</a> Nature. 574 (7778), 418-422 (2019).</li><li>Notaras, 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=Schizophrenia+is+defined+by+cell-specific+neuropathology+and+multiple+neurodevelopmental+mechanisms+in+patient-derived+cerebral+organoids.">Schizophrenia is defined by cell-specific neuropathology and multiple neurodevelopmental mechanisms in patient-derived cerebral organoids.</a> Mol Psychiatry. 27 (3), 1416-1434 (2022).</li><li>Rybak-Wolf, 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=Modelling+viral+encephalitis+caused+by+herpes+simplex+virus+1+infection+in+cerebral+organoids.">Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids.</a> Nat Microbiol. 8 (7), 1252-1266 (2023).</li><li>Bock, C., 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+organoid+cell+atlas.">The organoid cell atlas.</a> Nat Biotechnol. 39 (1), 13-17 (2021).</li><li>Brazovskaja, A., Treutlein, B., Camp, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+single-cell+transcriptomics+on+organoids.">High-throughput single-cell transcriptomics on organoids.</a> Cur Opinion Biotechnol. 55, 167-171 (2019).</li><li>Velasco, 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=Individual+brain+organoids+reproducibly+form+cell+diversity+of+the+human+cerebral+cortex.">Individual brain organoids reproducibly form cell diversity of the human cerebral cortex.</a> Nature. 570, 523-527 (2019).</li><li>Uzquiano, 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=Proper+acquisition+of+cell+class+identity+in+organoids+allows+definition+of+fate+specification+programs+of+the+human+cerebral+cortex.">Proper acquisition of cell class identity in organoids allows definition of fate specification programs of the human cerebral cortex.</a> Cell. 185 (20), 3770-3788.e27 (2022).</li><li>Mattei, 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=Enzymatic+dissociation+induces+transcriptional+and+proteotype+bias+in+brain+cell+populations.">Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations.</a> Int J Mol Sci. 21 (21), 7944 (2020).</li><li>Van Den Brink, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+sequencing+reveals+dissociation-induced+gene+expression+in+tissue+subpopulations.">Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations.</a> Nat Methods. 14 (10), 935-936 (2017).</li><li>Slyper, 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=A+single-cell+and+single-nucleus+RNA-Seq+toolbox+for+fresh+and+frozen+human+tumors.">A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.</a> Nat Med. 26 (5), 792-802 (2020).</li><li>Santos, M. 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=Extraction+and+sequencing+of+single+nuclei+from+murine+skeletal+muscles.">Extraction and sequencing of single nuclei from murine skeletal muscles.</a> STAR Protoc. 2 (3), 100694 (2021).</li><li>Fleck, J. 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=Inferring+and+perturbing+cell+fate+regulomes+in+human+cerebral+organoids.">Inferring and perturbing cell fate regulomes in human cerebral organoids.</a> Nature. 621 (7978), 365-372 (2021).</li><li>Martins-Costa, C., 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=Morphogenesis+and+development+of+human+telencephalic+organoids+in+the+absence+and+presence+of+exogenous+extracellular+matrix.">Morphogenesis and development of human telencephalic organoids in the absence and presence of exogenous extracellular matrix.</a> EMBO J. 42 (22), e113213 (2023).</li><li>Hao, Y., 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=Integrated+analysis+of+multimodal+single-cell+data.">Integrated analysis of multimodal single-cell data.</a> Cell. 184 (13), 3573-3587.e29 (2021).</li><li>Choe, M. 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=A+simple+method+to+improve+the+quality+and+yield+of+human+pluripotent+stem+cell-derived+cerebral+organoids.">A simple method to improve the quality and yield of human pluripotent stem cell-derived cerebral organoids.</a> Heliyon. 7 (6), e07350 (2021).</li><li>Giandomenico, S. 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=Cerebral+organoids+at+the+air-liquid+interface+generate+diverse+nerve+tracts+with+functional+output.">Cerebral organoids at the air-liquid interface generate diverse nerve tracts with functional output.</a> Nat Neurosci. 22 (4), 669-679 (2019).</li><li>Denisenko, E., 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=Systematic+assessment+of+tissue+dissociation+and+storage+biases+in+single-cell+and+single-nucleus+RNA-seq+workflows.">Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows.</a> Genome Biol. 21 (1), 130 (2020).</li><li>Wen, F., Tang, X., Xu, L., Qu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+single-nucleus+and+single-cell+transcriptomes+in+hepatocellular+carcinoma+tissue.">Comparison of single-nucleus and single-cell transcriptomes in hepatocellular carcinoma tissue.</a> Mol Med Rep. 26 (5), 339 (2022).</li><li>Alles, 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=Cell+fixation+and+preservation+for+droplet-based+single-cell+transcriptomics.">Cell fixation and preservation for droplet-based single-cell transcriptomics.</a> BMC Biol. 15 (1), 44 (2017).</li></ol>

Transcriptomic analysis of single cells and single nuclei has emerged as a pivotal tool for understanding gene regulatory mechanisms within complex tissues. Both methods enable transcriptome studies of brain organoids. To ensure an overall successful experiment, the quality of the starting material is of high relevance. Therefore, it is necessary to cut the organoids regularly to prevent the formation of a necrotic core26. It is also possible to eliminate this issue with an Air-Liquid Interface cu...

Over the last years, organoid technologies have emerged as a promising tool to culture organ-like tissues1,2,3. Especially for organs that cannot be easily accessed, such as the human brain, organoids offer the opportunity to gain insights into&#160;development and disease manifestation4. As such, brain organoids have been widely used as an experimental model to investigate various human brain disorders, including developmental, psychiatric, or even neurodegenerative diseases4,5,6.
With the advent of single-cell transcriptome profiling technologies, primary human tissues and complex in vitro models could be studied with an unprecedented level of granularity, providing mechanistic insights into gene expression changes on the level of cell subpopulations in health and disease and informing about new putative therapeutic targets7,8,9. The organoid field has progressed by utilizing single-cell transcriptome profiling to assess cellular composition, reproducibility and the fidelity of brain organoid technologies10,11,12. Single-cell RNA-sequencing (scRNA-seq) enabled cell classification and the identification of genetic dysregulation in diseased organoids13,14. Importantly, it is the complexity of organoid tissues that necessitates the implementation of techniques that enable the profiling of individual cells. Characterization of organoids using methods such as bulk transcriptome profiling (bulk RNA sequencing) leads to masked cellular heterogeneity and gene expression profiles which are averaged across all types of cells within the complex tissue, ultimately limiting our understanding of ongoing processes during organoid development in health and disease15,16,17. As scRNA-seq methods continue to advance, an increasing number of atlases are being created, exemplified by resources like the Allen Brain Atlas or the Single cell atlas of human brain organoids by Uzquiano et al.18.
Accomplishing successful scRNA-seq from brain organoids relies on effective isolation and capture of intact cells. As the dissociation of brain organoids to obtain individual cells is based on enzymatic digestion, it can influence gene expression patterns by inducing stress and cell damage19,20. Hence, the dissociation of the tissue into individual cells is the most crucial step. An alternative approach is single-nucleus RNA sequencing (snRNA-seq), which facilitates the enzyme-free extraction of nuclei from both, fresh and frozen, tissue21,22. However, the isolation of nuclei from a tissue poses other challenges such as the enrichment of cell types of interest and the low RNA content of nuclei in comparison to cells.
Transcriptome studies of brain organoids are commonly conducted using scRNA-seq10,18,23. However, the isolation of single nuclei might provide an orthogonal and supplemental method to investigate the transcriptomic profile of organoids. Here, we introduce a toolbox for scRNA- and snRNA-seq for brain organoids and discuss the critical points for obtaining the best quality sequencing data.

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			<td>1,4-DITHIO-DL-THREIT-LSG., F. D. MOL.-BIOL., ~1 M IN H2O (DTT)</td>
			<td>Sigma</td>
			<td>&#160;43816-10ML</td>
			<td></td>
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			<td>1.5 ml DNA low binding tubes&#160;</td>
			<td>VWR</td>
			<td>525-0130</td>
			<td>microcentrifuge tube</td>
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			<td>10x Cellranger pipeline&#160;</td>
			<td></td>
			<td></td>
			<td>analysis pipline</td>
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			<td>15 ml Falcon</td>
			<td>Falcon</td>
			<td></td>
			<td>Centrifuge tube</td>
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			<td>2-Mercaptoethanol (BME)</td>
			<td>Life Technologies</td>
			<td>21985023</td>
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			<td>50 ml Falcon</td>
			<td>Falcon</td>
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			<td>Centrifuge tube</td>
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			<td>A83-01</td>
			<td>Bio Technologies</td>
			<td>379762</td>
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			<td>Antibiotic/Antimycotic Solution (100X)</td>
			<td>Life Technologies</td>
			<td>15240062</td>
			<td></td>
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			<td>B-27 Plus Supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td></td>
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			<td>B-27 Supplement without vitamin A</td>
			<td>Life Technologies</td>
			<td>12587010</td>
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			<td>Bovine serum albumin, fatty acid free (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A8806-5G&#160;</td>
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			<td>cAMP</td>
			<td>Biogems</td>
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			<td>cAMP</td>
			<td>Biogems</td>
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			<td>C-CHIP NEUBAUER IMPROVED</td>
			<td>VWR</td>
			<td>DHC-N01</td>
			<td></td>
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			<td>Cell strainer 40 &#181;m</td>
			<td>Neolab</td>
			<td>352340</td>
			<td></td>
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			<td>Cell strainer 70 &#181;m (white) Nylon</td>
			<td>Sigma</td>
			<td>CLS431751-50EA</td>
			<td></td>
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			<td>Chromium Controller &#38; Next GEM Accessory Kit</td>
			<td>10X Genomics</td>
			<td>1000204</td>
			<td></td>
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			<td>Chromium Next GEM Chip G Single Cell Kit, 16 rxns</td>
			<td>10X Genomics</td>
			<td>1000127</td>
			<td></td>
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			<td>Chromium Next GEM Single Cell 3&#39; Kit v3.1</td>
			<td>10X Genomics</td>
			<td>1000268</td>
			<td></td>
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			<td>Complete,&#160; EDTA-free Protease Inhibitor Cocktaill</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
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			<td>DAPI</td>
			<td>MERCK Chemicals</td>
			<td>0000001722</td>
			<td></td>
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			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11320074</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce tissue grinder set 2 mL complete</td>
			<td>Sigma Aldrich</td>
			<td>10536355</td>
			<td></td>
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		<tr>
			<td>Essential E8 Flex Medium</td>
			<td>Life Technologies</td>
			<td>A2858501</td>
			<td></td>
		</tr>
		<tr>
			<td>EVE Cell Counting Slides</td>
			<td>VWR</td>
			<td>EVS-050 ( 734-2676)</td>
			<td></td>
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			<td>Foetal bovine serum tetracycline free (FBS)</td>
			<td>PAN Biotech</td>
			<td>P30-3602</td>
			<td></td>
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			<td>Geltrex LDEV-Free (coating)</td>
			<td>Life Technologies</td>
			<td>A1413302&#160;</td>
			<td></td>
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			<td>gentleMACS</td>
			<td>Miltenyi Biotec</td>
			<td></td>
			<td>dissociation maschine</td>
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			<td>GlutaMAX supplements</td>
			<td>Life Technologies</td>
			<td>35050038</td>
			<td></td>
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			<td>Heparin sodium cell culture tested</td>
			<td>Sigma</td>
			<td>H3149-10KU</td>
			<td></td>
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			<td>human recombinant BDNF</td>
			<td>StemCell Technologies</td>
			<td>78005.3</td>
			<td></td>
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			<td>human recombinant GDNF</td>
			<td>StemCell Technologies</td>
			<td>78058.3</td>
			<td></td>
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			<td>Insulin Solution Human</td>
			<td>Sigma Aldrich</td>
			<td>I2643-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout serum replacement</td>
			<td>Life Technologies</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189 Hydrochloride 98%</td>
			<td>Sigma Aldrich</td>
			<td>130-106-540</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acid (100x)</td>
			<td>Sigma Aldrich</td>
			<td>M7145-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 Magnesium Chloride (1M) RNAse free</td>
			<td>Thermo Scientific</td>
			<td>AM9530G</td>
			<td></td>
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			<td>mTeSR Plus</td>
			<td>StemCell Technologies</td>
			<td>100-0276</td>
			<td>stem cell medium</td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>StemCell Technologies</td>
			<td>85850</td>
			<td>stem cell medium</td>
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		<tr>
			<td>N2 Supplement&#160;</td>
			<td>StemCell Technologies</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural Tissue Dissociation Kit</td>
			<td>Miltenyi Biotec B.V. &#38; Co. KG</td>
			<td>130-092-628</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Plus</td>
			<td>Life Technologies</td>
			<td>A3582901</td>
			<td></td>
		</tr>
		<tr>
			<td>NextSeq500 system</td>
			<td>Illumina</td>
			<td></td>
			<td>Sequencer</td>
		</tr>
		<tr>
			<td>NP-40 Surfact-Amps Detergent Solution</td>
			<td>Life Technologies</td>
			<td>28324</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Dulbecco&#8217;s</td>
			<td>Invitrogen</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>PenStrep (Penicillin - Streptomycin)</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Th. Geyer</td>
			<td>10668276</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic (R) F-127</td>
			<td>Sigma Aldrich</td>
			<td>P2443-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>RiboLock RNase Inhibitor</td>
			<td>Life Technologies&#160;</td>
			<td>EO0382</td>
			<td></td>
		</tr>
		<tr>
			<td>Rock Inhibitor (Y-27632 dihydrochloride) SB</td>
			<td>Biomol</td>
			<td>Cay10005583-10</td>
			<td></td>
		</tr>
		<tr>
			<td>SB 431542&#160;</td>
			<td>Biogems</td>
			<td>3014193</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride NaCl (5M), RNase-free-100 mL</td>
			<td>Invitrogen</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>StemFlex Medium</td>
			<td>Thermo Scientific</td>
			<td>A3349401</td>
			<td>stem cell medium</td>
		</tr>
		<tr>
			<td>StemMACS iPS-Brew XF</td>
			<td>Miltenyi Biotec</td>
			<td>130-104-368</td>
			<td>stem cell medium</td>
		</tr>
		<tr>
			<td>TC-Platte 96 Well, round bottom</td>
			<td>Sarstedt</td>
			<td>83.3925.500</td>
			<td></td>
		</tr>
		<tr>
			<td>TISSUi006-A</td>
			<td>TissUse GmbH</td>
			<td>https://hpscreg.eu/cell-line/TISSUi006-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>T8154-20ml</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme, no phenol red</td>
			<td>Life Technologies</td>
			<td>12604013</td>
			<td>Trypsin-based reagent</td>
		</tr>
		<tr>
			<td>UltraPure 1M Tris-HCl Buffer, pH 7.5</td>
			<td>Life Technologies</td>
			<td>15567027</td>
			<td></td>
		</tr>
		<tr>
			<td>XAV939</td>
			<td>Enzo Life sciences</td>
			<td>BML-WN100-0005</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and downstream analysis of single-cell and single-nuclei transcriptomes in brain organoids

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene expression profiles of individual cells, allowing the capture of cellular diversity. In order to overcome limitations posed by difficult-to-isolate cell types, an alternative approach aiming at recovering single nuclei instead of intact cells can be utilized for sequencing, making transcriptome profiling of individual cells universally applicable. These techniques have become a cornerstone in the study of brain organoids, establishing them as models of the developing human brain. Leveraging the potential of single-cell and single-nucleus transcriptomics in brain organoid research, this protocol presents a step-by-step guide encompassing key procedures such as organoid dissociation, single-cell or nuclei isolation, library preparation&#160;and sequencing. By implementing these alternative approaches, researchers can obtain high-quality datasets, enabling the identification of neuronal and non-neuronal cell types, gene expression profiles, and cell lineage trajectories. This facilitates comprehensive investigations into cellular processes and molecular mechanisms shaping brain development.

Author Spotlight: Integrating Organoid Models with Single-Cell and Spatial Transcriptomics Technologies

Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids

To investigate cell type composition of brain organoids using scRNA-seq and snRNA-seq, brain organoids were harvested after 30 days of culture as organoids at this stage already exhibit neuroepithelial loops consisting of progenitors surrounded by intermediate progenitors and early stage neurons4,18. Monitoring the quality of the organoids throughout growth and culturing is essential for obtaining reliable single-cell and single-nucleus data.
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Watch this Scientific Journal Video about Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids at JoVE.com

Here, we introduce a comprehensive protocol for the generation and downstream analysis of human brain organoids using single-cell and single-nucleus RNA sequencing.

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene ...