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 different companies (Table 1). The generation of forebrain organoids is highly dependent on high quality iPSCs and a confluence of 60%-70% prior to starting the protocol. Here we used a commercially available cell line (see Table of Materials).
<ol>
	<li>Day 0: Embryoid body formation
	<ol>
		<li>For treatment of 96-well plate with pluronic solution, add 80 &#181;L of sterile filtered 2% pluronic solution per well of a 96-well U-bottom plate. Incubate for at least 15 min at room temperature (RT) or 37 &#176;C. 
		NOTE: Pluronic treated plates can be stored at 4 &#176;C for up to 2 weeks and can be used directly without any washing steps for embryoid body formation.</li>
		<li>Remove pluronic solution from each well using an aspirator or a multichannel pipette before seeding cells.</li>
		<li>Before starting, prepare the following reagents for one 96-well plate: 15 mL centrifuge tube with 5 mL of pre-warmed DMEM: F12 medium; 11 mL of stem cell medium supplemented with 50 &#181;M Y-27632, pluronic-treated plate (as described above).</li>
		<li>Aspirate media from one well of a 6-well plate of 60-70% confluent iPSCs. Wash cells once with PBS. Add 500 &#181;L of trypsin-based reagent and incubate for 2-6 min at 37 &#176;C. 
		NOTE: Incubation time for proper detachment of cells with trypsin-based reagent depends on cell density and the cell matrix-adhesive properties of each iPSC line. To avoid over digestion, it is advised to examine cell morphology after 2 min of incubation with trypsin-based reagent using a bright field microscope.</li>
		<li>Detach cells using a 1000 &#181;L pipette and transfer into the previously prepared 15 mL centrifuge tube with DMEM:F12 medium to stop dissociation.</li>
		<li>Centrifuge cell suspension for 3 min at 300 x g. Aspirate supernatant and keep the cell pellet. Resuspend the cells in 1 mL of stem cell medium supplemented with 50 &#181;M Y-27632.</li>
		<li>Count cells using a cell counter and add desired number of cells to the stem cell media supplemented with 50 &#181;M Y-27632. The generation of a single embryoid body requires 3,000-12,000 cells depending on the cell line.</li>
		<li>Transfer the cell suspension to a multichannel reagent reservoir using a 10 mL pipette. Using a multichannel pipette, plate 100 &#181;L/well of the cell suspension into the previously pluronic-treated 96-well plate.</li>
		<li>Place 96-well plate in an incubator at 37 &#176;C, 5% O2 and 5% CO2. To avoid any disturbance during embryoid body formation, refrain from moving the plate.</li>
	</ol>
	</li>
	<li>Day 2: Embryoid body formation examination
	<ol>
		<li>Examine embryoid body formation using a 10x objective of a bright field microscope. Embryoid bodies should display clear edges and minimal cell death.</li>
		<li>Aspirate as much medium as possible and replace with 100 &#181;L/well of stem cell medium. (Critical) Embryoid bodies are easily removed from the well during medium exchange. Pay attention to not remove them, therefore monitor medium after aspiration.</li>
	</ol>
	</li>
	<li>Day 3: Neuroectoderm induction
	<ol>
		<li>Aspirate as much medium as possible and replace with 120 &#181;L/well of induction medium and place plate at 37 &#176;C, 20% O2 and 5% CO2.</li>
	</ol>
	</li>
	<li>Day 6: Embedding
	<ol>
		<li>Depending on the iPSC line, 3 or 4 days are required to induce neuroectoderm emergence. Monitor embryoid bodies regularly. Once the edges turn bright, embryoid bodies are ready for embedding. Use one of the two different methods that can be used for embryoid body embedding as described below24.</li>
		<li>Embedding method 1: Cookie embedding
		<ol>
			<li>Thaw an aliquot of undiluted extracellular matrix gel on ice for 1-2 h. (Critical) Always keep extracellular matrix gel on ice to prevent it from solidifying. Prepare aliquots in advance to minimize thawing time and repeated thawing cycles.</li>
			<li>Cut the tip of a 200 &#181;L pipette using claw nippers and collect 16-32 embryoid bodies in one 1.5 mL microcentrifuge tube.</li>
			<li>Transfer the embryoid bodies in 67 &#181;L of the media into a new 1.5 mL microcentrifuge tube. Add 100 &#181;L of extracellular matrix gel using a cut 200 &#181;L pipette tip and mix gently.</li>
			<li>Evenly distribute the embryoid bodies in a 6 cm dish and place the plate for 5-10 min to allow the extracellular matrix gel to solidify.</li>
			<li>Add about 4 mL of differentiation media and incubate at 37 &#176;C, 20% O2 and 5% CO2. On the next day, place the dish on an orbital shaker (84 rpm). Ensure that all embryoid bodies detach from the bottom. If not, use a cut pipette tip and media to gently detach them.</li>
		</ol>
		</li>
		<li>Embedding method 2: Liquid embedding
		<ol>
			<li>Thaw an aliquot of undiluted extracellular matrix gel on ice for 1-2 h and place differentiation media on ice. (Critical) Media must be ice-cold when adding extracellular matrix gel.</li>
			<li>Once the medium is cold, add extracellular matrix gel to the final concentration of 2%. Cut the tip of a 200 &#181;L pipette using claw nippers and transfer up to 24 embryoid bodies to one well of a 6-well plate.</li>
			<li>Remove all excess media and add about 4 mL of 2% extracellular matrix gel/differentiation media into one well of a 6-well plate. Immediately place the plate on the orbital shaker (84 rpm).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Day 9-20: Perform a full media exchange using differentiation media every 3-4 days.</li>
	<li>Day 21-30: Transfer organoids into maturation media and perform full media exchanges every 3-4 days.</li>
	<li>Dissociation of organoids: For single nuclei and cell dissociation, use organoids of high quality. To avoid excess amounts of cell death, regularly cut the organoids to prevent the build-up of a necrotic core and allow them to recover for 2 weeks before dissociating them for further analysis.</li>
</ol>
2. Derivation of single cell from organoids
NOTE: Single cell dissociation is performed using the Neural Tissue Dissociation Kit (Table 2), which uses mechanical and enzymatic dissociation. Here we describe a manual mechanical dissociation. As an alternative, a dissociation machine can be used.
<ol>
	<li>Prepare STOP solution and 0.04% BSA on ice. Prepare enzyme mix 1 and 2 and 0.04% BSA in PBS and place on ice.</li>
	<li>Collect up to 5 organoids in one well of a 6-well plate and aspirate excess media and wash once with PBS to remove culture media. Place organoids in the middle of the well and using a scalpel, mince organoids thoroughly.</li>
	<li>Add enzyme mix 1 and place at 37 &#176;C, 20% O2 and 5% CO2, shaking at 90 rpm for 10-15 min.</li>
	<li>Using a 1000 &#181;L pipette tip, resuspend organoids by pipetting up to 10x. Add enzyme mix 2 and mix it well by pipetting using a 1000 &#181;L pipette tip. Place at 37 &#176;C, 20% O2 and 5% CO2, shaking at 90 rpm for 10-15 min.</li>
	<li>Stop the dissociation process by resuspending the cell solution in 10 mL of cold STOP solution. Filter cell solution using a 70 &#181;M cell strainer. Centrifuge the filtered cell solution for 10 min at 300 x g at 4 &#176;C to form a pellet.</li>
	<li>Aspirate media, leaving a cell pellet and resuspend cells in 4 mL of cold 0.04% BSA. Filter cell solution using a 40 &#181;M cell strainer and count cells using a cell counter.</li>
	<li>Centrifuge the cell solution for 10 min at 300 x g at 4 &#176;C. Aspirate BSA solution, leaving a cell pellet. Resuspend cells according to a microfluidics-based-snRNA-seq kit manual in NSB+ medium.</li>
</ol>
3. Isolation of single nuclei from organoids
<ol>
	<li>Transfer organoids into chilled PBS and cut each organoid into 4 pieces. Wash organoids 2x with chilled PBS.</li>
	<li>Cut with scissors the pipette tip of a 1000 &#181;L pipette and transfer organoid pieces into a 2 mL douncer. Aspirate PBS completely and add 1 mL of NP40 lysis buffer.</li>
	<li>Dounce 3x with dounce pestle A and 3x with dounce pestle B. Transfer suspension into a 15 mL centrifuge tube. Wash douncer with an additional 1 mL of NP40 lysis buffer and transfer into a 15 mL centrifuge tube. Incubate for 5 min at RT and subsequently centrifuge suspension for 5 min at 500 x g.</li>
	<li>Aspirate supernatant leaving 50 &#181;L of supernatant behind. Add 1 mL of NSB+ without disturbing the pellet. Resuspend after 5 min of incubation.</li>
	<li>Layer suspension on top of a Percoll gradient (bottom layer: 1 mL of NSB + and 250 &#181;L of Percoll, middle layer: 1 mL of NSB+ and 188 &#181;L of Percoll, top layer: 1 mL of NSB+ and 125 &#181;L of Percoll). (Critical) Perform layering very carefully to avoid layer mixing.</li>
	<li>Centrifuge for 5 min at 500 x g at 4 &#176;C, aspirate supernatant and resuspend in NSB+. Filter nuclei solution using a 40 &#181;M cell strainer.</li>
	<li>Stain and count nuclei using DAPI and a Neubauer Chamber. Resuspend nuclei according to a microfluidics-based scRNA-seq kit manual.</li>
</ol>
4. Library preparation and sequencing
<ol>
	<li>Load 10,000 cells and nuclei into a microfluidics system and generate the library according to the manual of microfluidics based scRNA-seq kit (Table of Materials).</li>
	<li>Sequence the libraries with an estimated 6 x 108 reads per snRNA-seq library and 1.6 x 108 reads per scRNA-seq library, resulting in a sequencing saturation of 87% for the snRNA-seq dataset and 36% sequencing saturation for the scRNA-seq dataset.</li>
</ol>
5. Analysis
<ol>
	<li>Carry out analysis of sequencing using a data analysis pipeline for scRNA-seq and snRNA-seq and align against the human GRCh38 genome. Subject the count matrix generated by this pipeline to further analysis using the R-package Seurat (Version 4.1.1)25.</li>
	<li>Create the Seurat object by considering genes that were represented in at least 5 cells and incorporating cells that contained at least 300 genes. Perform additional quality control filtering by retaining cells that contained more than 1000 genes but less than 4000 genes for the scRNA-seq dataset and more than 800, but less than 4000 genes per nuclei for the snRNA-seq dataset. Exclude datasets cells and nuclei exceeding 5% mitochondrial reads.</li>
	<li>To mitigate batch effects between both methods, integrate both filtered datasets, normalize and visualize via Uniform Manifold Approximation and Projection (UMAP). Omit cluster 1 which displays low unique molecular identifier (UMI) and gene counts. Afterwards, for the clusters assign cell identities based on known marker genes and the 30-day-old organoid dataset from Ana Uzquiano et. al. (Supplementary Coding File 1)18.</li>
</ol>

Single Cell and Nuclei Extraction from Human Brain Organoids

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The authors declare no competing financial interests.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<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>
		</tr>
		<tr>
			<td>1.5 ml DNA low binding tubes&#160;</td>
			<td>VWR</td>
			<td>525-0130</td>
			<td>microcentrifuge tube</td>
		</tr>
		<tr>
			<td>10x Cellranger pipeline&#160;</td>
			<td></td>
			<td></td>
			<td>analysis pipline</td>
		</tr>
		<tr>
			<td>15 ml Falcon</td>
			<td>Falcon</td>
			<td></td>
			<td>Centrifuge tube</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (BME)</td>
			<td>Life Technologies</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Falcon</td>
			<td>Falcon</td>
			<td></td>
			<td>Centrifuge tube</td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Bio Technologies</td>
			<td>379762</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic/Antimycotic Solution (100X)</td>
			<td>Life Technologies</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Plus Supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement without vitamin A</td>
			<td>Life Technologies</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin, fatty acid free (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A8806-5G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>Biogems</td>
			<td>6099240</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>Biogems</td>
			<td>6099240</td>
			<td></td>
		</tr>
		<tr>
			<td>C-CHIP NEUBAUER IMPROVED</td>
			<td>VWR</td>
			<td>DHC-N01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 40 &#181;m</td>
			<td>Neolab</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 70 &#181;m (white) Nylon</td>
			<td>Sigma</td>
			<td>CLS431751-50EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Controller &#38; Next GEM Accessory Kit</td>
			<td>10X Genomics</td>
			<td>1000204</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Next GEM Chip G Single Cell Kit, 16 rxns</td>
			<td>10X Genomics</td>
			<td>1000127</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Next GEM Single Cell 3&#39; Kit v3.1</td>
			<td>10X Genomics</td>
			<td>1000268</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete,&#160; EDTA-free Protease Inhibitor Cocktaill</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>MERCK Chemicals</td>
			<td>0000001722</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Foetal bovine serum tetracycline free (FBS)</td>
			<td>PAN Biotech</td>
			<td>P30-3602</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex LDEV-Free (coating)</td>
			<td>Life Technologies</td>
			<td>A1413302&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS</td>
			<td>Miltenyi Biotec</td>
			<td></td>
			<td>dissociation maschine</td>
		</tr>
		<tr>
			<td>GlutaMAX supplements</td>
			<td>Life Technologies</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium cell culture tested</td>
			<td>Sigma</td>
			<td>H3149-10KU</td>
			<td></td>
		</tr>
		<tr>
			<td>human recombinant BDNF</td>
			<td>StemCell Technologies</td>
			<td>78005.3</td>
			<td></td>
		</tr>
		<tr>
			<td>human recombinant GDNF</td>
			<td>StemCell Technologies</td>
			<td>78058.3</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<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>
		</tr>
		<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

We are developing and optimizing organoid models in order to apply them for disease modeling. Our goal is to understand the molecular mechanism underlying the disease development and progression, and we are working in close collaboration with Nikolaus Rajewsky Lab, who is developing and applying the single-cell and spatial technologies in order to understand how the RNA regulate the gene expression in health and disease. In recent years, brain organoids have emerged as experimental and translational platforms for studying patient-specific diseases.Therefore, it's not unsurprising that most recent developments aimed at improving the complexity of brain organoid models. For instance, we are now able to generate brain region-specific organoids and also use brain organoids for multi-organoid assemblies. Also, various methods have been implemented to introduce vascularization and immune cells into brain organoids.Currently, one of the most research-advancing technologies that we're using in this lab is spatial transcriptomics, which allows the assessment of gene expression within intact tissue. This is crucial in understanding how gene expression profiles of different cell types vary in different regions within one tissue. Another recent advancement that has been made is single-cell multiomics, which allows the analysis of multiple modules of a single cell, which gives a more holistic approach in analyzing these cells.We employ brain organoids and single-cell and spatial transcriptomics methods to understand underlying mechanisms of human diseases such as Leigh syndrome and herpes simplex encephalitis. Furthermore, we developed high-resolution and cost-effective spatial transcriptomics methods called Open-ST. We compared and optimized the single-cell and single-nucleus RNA sequencing method to understand how well they perform regarding cell type recovery in brain organoid tissue.

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 ...

generation-downstream-analysis-single-cell-single-nuclei

Research

JoVE Journal

Developmental Biology

Isolation of Single Cells from Human Brain Organoids for Single-Cell RNA Sequencing

To begin, collect up to five human brain organoids generated from induced pluripotent stem cells in one well of a six well plate. Aspirate excess culture media, and wash the organoids once with PBS. Using a scalpel, mince the organoids thoroughly.Add enzyme mix one to the minced organoids, and incubate at 37 degrees Celsius with 20%oxygen and 5%carbon dioxide at 90 RPM for 10 to 15 minutes. Mix the organoid suspension using a 1000 microliter pipette tip. Then add enzyme mix two, and incubate at 37 degrees Celsius for 10 to 15 minutes.Stop dissociation by adding 10 milliliters of stop solution. Filter the cell suspension on a 70-micron cell strainer. Centrifuge the filtered cell suspension at 300 G for 10 minutes at four degrees Celsius.Aspirate the supernatant, and re-suspend the pellet in four milliliters of cold 0.04%BSA. Now filter the cell suspension on a 40-micron cell strainer, and count the cells on a cell counter. Centrifuge the required amount of cell suspension, aspirate the supernatant, and re-suspend the pellet in NSB plus medium according to a microfluidics-based scRNA-Seq kit manual.

Isolation of Single Nuclei from Human Brain Organoids for Single-Nucleus RNA Sequencing

To begin, transfer four human brain organoids generated from induced pluripotent stem cells into a six-well plate containing chilled PBS. Cut each organoid into four pieces. Using scissors, cut the tip of a 1, 000 microliter pipette tip and use it to transfer organoid pieces into a two milliliter douncer.Aspirate PBS completely and add one milliliter of NP-40 lysis buffer. Dounce the organoids three times with dounce pestle A followed by pestle B.Transfer the suspension into a 15 milliliter centrifuge tube. Wash the douncer with one milliliter of lysis buffer and transfer the solution to a centrifuge tube.After five minutes of incubation at room temperature, centrifuge the organoid suspension at 500 G for five minutes. Aspirate the supernatant leaving 50 microliters in the tube. Add one milliliter of NSB Plus medium into the tube.And after five minutes, mix to resuspend the pellet. After preparing a Percoll gradient in the tube, layer the organoid suspension on top of it. Next, centrifuge the layered organoid suspension at 500 G for five minutes at four degrees Celsius.Aspirate the supernatant and resuspend the pellet in NSB Plus medium. Filter the nuclei solution on a 40 micron cell strainer. Then, stain the nuclei using DAPI and count them in a Neubauer chamber.Centrifuge the required amount of nuclei solution and resuspend the pellet in NSB Plus medium according to a microfluidics based snRNA-seq kit manual.

1.5K Views.  Max Delbrück Center for Molecular Medicine (MDC).  Here, we introduce a comprehensive protocol for the generation and downstream analysis of human brain organoids using single-cell and single-nucleus RNA sequencing.

Single Cell and Nuclei Extraction from Human Brain Organoids

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