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* These authors contributed equally
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 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 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.
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 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,
The described protocol is performed in a biosafety level 1 laboratory of the Max Delbrück 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.......
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.
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.......
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.
....Name | Company | Catalog Number | Comments |
1,4-DITHIO-DL-THREIT-LSG., F. D. MOL.-BIOL., ~1 M IN H2O (DTT) | Sigma | 43816-10ML | |
1.5 ml DNA low binding tubes | VWR | 525-0130 | microcentrifuge tube |
10x Cellranger pipeline | analysis pipline | ||
15 ml Falcon | Falcon | Centrifuge tube | |
2-Mercaptoethanol (BME) | Life Technologies | 21985023 | |
50 ml Falcon | Falcon | Centrifuge tube | |
A83-01 | Bio Technologies | 379762 | |
Antibiotic/Antimycotic Solution (100X) | Life Technologies | 15240062 | |
B-27 Plus Supplement | Life Technologies | 17504044 | |
B-27 Supplement without vitamin A | Life Technologies | 12587010 | |
Bovine serum albumin, fatty acid free (BSA) | Sigma Aldrich | A8806-5G | |
cAMP | Biogems | 6099240 | |
cAMP | Biogems | 6099240 | |
C-CHIP NEUBAUER IMPROVED | VWR | DHC-N01 | |
Cell strainer 40 µm | Neolab | 352340 | |
Cell strainer 70 µm (white) Nylon | Sigma | CLS431751-50EA | |
Chromium Controller & Next GEM Accessory Kit | 10X Genomics | 1000204 | |
Chromium Next GEM Chip G Single Cell Kit, 16 rxns | 10X Genomics | 1000127 | |
Chromium Next GEM Single Cell 3' Kit v3.1 | 10X Genomics | 1000268 | |
Complete, EDTA-free Protease Inhibitor Cocktaill | Roche | 11873580001 | |
DAPI | MERCK Chemicals | 0000001722 | |
DMEM/F12 | Life Technologies | 11320074 | |
Dounce tissue grinder set 2 mL complete | Sigma Aldrich | 10536355 | |
Essential E8 Flex Medium | Life Technologies | A2858501 | |
EVE Cell Counting Slides | VWR | EVS-050 ( 734-2676) | |
Foetal bovine serum tetracycline free (FBS) | PAN Biotech | P30-3602 | |
Geltrex LDEV-Free (coating) | Life Technologies | A1413302 | |
gentleMACS | Miltenyi Biotec | dissociation maschine | |
GlutaMAX supplements | Life Technologies | 35050038 | |
Heparin sodium cell culture tested | Sigma | H3149-10KU | |
human recombinant BDNF | StemCell Technologies | 78005.3 | |
human recombinant GDNF | StemCell Technologies | 78058.3 | |
Insulin Solution Human | Sigma Aldrich | I2643-25MG | |
Knockout serum replacement | Life Technologies | 10828028 | |
LDN193189 Hydrochloride 98% | Sigma Aldrich | 130-106-540 | |
MEM non-essential amino acid (100x) | Sigma Aldrich | M7145-100ml | |
MgCl2 Magnesium Chloride (1M) RNAse free | Thermo Scientific | AM9530G | |
mTeSR Plus | StemCell Technologies | 100-0276 | stem cell medium |
mTeSR1 | StemCell Technologies | 85850 | stem cell medium |
N2 Supplement | StemCell Technologies | 17502048 | |
Neural Tissue Dissociation Kit | Miltenyi Biotec B.V. & Co. KG | 130-092-628 | |
Neurobasal Plus | Life Technologies | A3582901 | |
NextSeq500 system | Illumina | Sequencer | |
NP-40 Surfact-Amps Detergent Solution | Life Technologies | 28324 | |
PBS Dulbecco’s | Invitrogen | 14190169 | |
PenStrep (Penicillin - Streptomycin) | Life Technologies | 15140122 | |
Percoll | Th. Geyer | 10668276 | |
Pluronic (R) F-127 | Sigma Aldrich | P2443-1KG | |
RiboLock RNase Inhibitor | Life Technologies | EO0382 | |
Rock Inhibitor (Y-27632 dihydrochloride) SB | Biomol | Cay10005583-10 | |
SB 431542 | Biogems | 3014193 | |
Sodium chloride NaCl (5M), RNase-free-100 mL | Invitrogen | AM9760G | |
StemFlex Medium | Thermo Scientific | A3349401 | stem cell medium |
StemMACS iPS-Brew XF | Miltenyi Biotec | 130-104-368 | stem cell medium |
TC-Platte 96 Well, round bottom | Sarstedt | 83.3925.500 | |
TISSUi006-A | TissUse GmbH | https://hpscreg.eu/cell-line/TISSUi006-A | |
Trypan Blue | T8154-20ml | Sigma | |
TrypLE Express Enzyme, no phenol red | Life Technologies | 12604013 | Trypsin-based reagent |
UltraPure 1M Tris-HCl Buffer, pH 7.5 | Life Technologies | 15567027 | |
XAV939 | Enzo Life sciences | BML-WN100-0005 |
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