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

Vi takker Valeria Fernandez-Vallone for de originale instruksjonene for Miltenyi Neural Dissociation-settet. Vi takker også Genomics Technology Platform til Max Delbrueck Centrum for å gi oppskriften på NP40-lysbufferen og verdifulle råd om å sette opp denne protokollen. Vi takker også Margareta Herzog og Alexandra Tschernycheff for laboratoriets organisatoriske støtte.

Den beskrevne protokollen utføres i et biosikkerhetsnivå 1-laboratorium ved Max Delbrück Center for Molecular Medicine (godkjenningsnummer: 138/08), i samsvar med kravene og i samsvar med EUs og nasjonale regler om etikk i forskning.
1. Avledning av organoider i forhjernen fra induserte pluripotente stamceller (iPSC)
MERK: Denne protokollen ble testet for flere forskjellige iPSC-linjer dyrket i en rekke stamcellemedier fra forskjellige selskaper (<...

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>

Transkriptomisk analyse av enkeltceller og enkeltkjerner har dukket opp som et sentralt verktøy for å forstå genreguleringsmekanismer i komplekse vev. Begge metodene muliggjør transkriptomstudier av hjerneorganoider. For å sikre et generelt vellykket eksperiment er kvaliteten på utgangsmaterialet av høy relevans. Derfor er det nødvendig å kutte organoidene regelmessig for å forhindre dannelse av en nekrotisk kjerne26. Det er også mulig å eliminere dette problemet med en Air-Liquid Inte...

I løpet av de siste årene har organoidteknologier dukket opp som et lovende verktøy for å dyrke organlignende vev 1,2,3. Spesielt for organer som ikke er lett tilgjengelige, for eksempel den menneskelige hjernen, gir organoider muligheten til å få innsikt i utvikling og sykdomsmanifestasjon4. Som sådan har hjerneorganoider blitt mye brukt som en eksperimentell modell for å undersøke ulike menneskelige hjernesykdommer, inkludert utviklingsmessige, psykiatriske eller til og med nevrodegenerative sykdommer 4,5,6.
Med bruken av encellede transkriptomprofileringsteknologier, kan primært humant vev og komplekse in vitro-modeller studeres med et enestående nivå av granularitet, noe som gir mekanistisk innsikt i genuttrykksendringer på nivået av cellesubpopulasjoner i helse og sykdom og informerer om nye antatte terapeutiske mål 7,8,9. Organoidfeltet har utviklet seg ved å bruke encellet transkriptomprofilering for å vurdere cellulær sammensetning, reproduserbarhet og troskapen til hjerneorganoidteknologier 10,11,12. Enkeltcellet RNA-sekvensering (scRNA-seq) muliggjorde celleklassifisering og identifisering av genetisk dysregulering i syke organoider13,14. Viktigere er det at det er kompleksiteten til organoidvev som nødvendiggjør implementering av teknikker som muliggjør profilering av individuelle celler. Karakterisering av organoider ved bruk av metoder som bulk transkriptomprofilering (bulk RNA-sekvensering) fører til maskerte cellulære heterogenitets- og genuttrykksprofiler som er gjennomsnittlige på tvers av alle typer celler i det komplekse vevet, noe som til slutt begrenser vår forståelse av pågående prosesser under organoidutvikling i helse og sykdom 15,16,17. Etter hvert som scRNA-seq-metodene fortsetter å utvikle seg, blir det laget et økende antall atlas, eksemplifisert av ressurser som Allen Brain Atlas eller Single cell atlas of human brain organoids av Uzquiano et al.18.
Å oppnå vellykket scRNA-seq fra hjerneorganoider er avhengig av effektiv isolering og fangst av intakte celler. Siden dissosiasjonen av hjerneorganoider for å oppnå individuelle celler er basert på enzymatisk fordøyelse, kan det påvirke genuttrykksmønstre ved å indusere stress og celleskade19,20. Derfor er dissosiasjonen av vevet i individuelle celler det mest avgjørende trinnet. En alternativ tilnærming er enkeltkjerne-RNA-sekvensering (snRNA-seq), som letter enzymfri ekstraksjon av kjerner fra både ferskt og frossent vev21,22. Isolering av kjerner fra et vev byr imidlertid på andre utfordringer som anrikning av celletyper av interesse og det lave RNA-innholdet i kjerner sammenlignet med celler.
Transkriptomstudier av hjerneorganoider utføres vanligvis ved bruk av scRNA-seq 10,18,23. Imidlertid kan isolering av enkeltkjerner gi en ortogonal og supplerende metode for å undersøke den transkriptomiske profilen til organoider. Her introduserer vi en verktøykasse for scRNA- og snRNA-seq for hjerneorganoider og diskuterer de kritiske punktene for å oppnå sekvenseringsdata av beste kvalitet.

<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

I løpet av det siste tiåret har encellet transkriptomikk utviklet seg betydelig og blitt en standard laboratoriemetode for samtidig analyse av genekspresjonsprofiler til individuelle celler, noe som tillater fangst av cellulært mangfold. For å overvinne begrensninger som utgjøres av celletyper som er vanskelige å isolere, kan en alternativ tilnærming som tar sikte på å gjenopprette enkeltkjerner i stedet for intakte celler brukes til sekvensering, noe som gjør transkriptomprofilering av individuelle celler universelt anvendelig. Disse teknikkene har blitt en hjørnestein i studiet av hjerneorganoider, og etablerer dem som modeller for den utviklende menneskelige hjernen. Ved å utnytte potensialet til enkeltcelle- og enkeltkjernetranskriptomikk i hjerneorganoidforskning, presenterer denne protokollen en trinn-for-trinn-veiledning som omfatter nøkkelprosedyrer som organoiddissosiasjon, encelle- eller kjerneisolasjon, bibliotekforberedelse og sekvensering. Ved å implementere disse alternative tilnærmingene kan forskere få datasett av høy kvalitet, som muliggjør identifisering av nevronale og ikke-nevronale celletyper, genuttrykksprofiler og celleavstamningsbaner. Dette legger til rette for omfattende undersøkelser av cellulære prosesser og molekylære mekanismer som former hjernens utvikling.

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.

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

Generering og nedstrømsanalyse av encellede og enkeltkjernede transkriptomer i hjerneorganoider

For å undersøke celletypesammensetningen til hjerneorganoider ved bruk av scRNA-seq og snRNA-seq, ble hjerneorganoider høstet etter 30 dagers kultur, da organoider på dette stadiet allerede viser nevroepitelsløyfer bestående av forfedre omgitt av mellomliggende forfedre og nevroner i tidlig stadium 4,18. Overvåking av kvaliteten på organoidene gjennom vekst og dyrking er avgjørende for å skaffe pålitelige encelle- og enkeltkjernedata.
<p class="jov...

Watch this Scientific Journal Video about Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids at JoVE.com

Her introduserer vi en omfattende protokoll for generering og nedstrømsanalyse av humane hjerneorganoider ved bruk av encelle- og enkeltkjerne-RNA-sekvensering.

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