Enkelt celle-RNA-sekvensering og analyse af humane bugspytkirtel-øer
Summary
Her præsenterer vi en protokol til at generere høj kvalitet, storstilet transkriptomer data af enkeltceller fra isolerede menneskelige pancreas Holme ved hjælp af en dråbe baseret mikrofluidisk enkelt celle RNA sekventering teknologi.
Abstract
Pancreasøer består af endokrine celler med karakteristiske hormon udtryks mønstre. De endokrine celler viser funktionelle forskelle som reaktion på normale og patologiske tilstande. Målet med denne protokol er at generere høj kvalitet, storstilet transkriptomer data for hver endokrine celletype med brug af en dråbe baseret mikrofluidisk enkelt celle RNA sekventering teknologi. Sådanne data kan udnyttes til at opbygge genekspressions profilen for hver endokrine celletype under normale eller specifikke forhold. Processen kræver omhyggelig håndtering, nøjagtig måling og streng kvalitetskontrol. I denne protokol beskriver vi detaljerede trin for humane pancreasislets dissociation, sekvensering og dataanalyse. De repræsentative resultater af ca. 20.000 humane enkelt-ø-celler viser, at protokollen er blevet gennemført korrekt.
Introduction
Pancreasislets frigive endokrine hormoner til at regulere blodsukkerniveauet. Fem endokrine celletyper, der adskiller sig funktionelt og morfologisk, er involveret i denne væsentlige rolle: α-celler producerer Glucagon, β-celler insulin, δ-celler somatostatin, PP celler Pankreatisk polypeptid, og ε-celler Ghrelin1. Genekspression profilering er en nyttig metode til at karakterisere de endokrine celler under normale eller specifikke forhold. Historisk set blev hele øen gen Expression profilering genereret ved hjælp af microarray og næste generations RNA-sekvensering2,3,4,5,6,7 , 8. selv om hele øen transkriptomet er informativt at identificere de orgel-specifikke udskrifter og sygdoms kandidat gener, det undlader at afdække den molekylære heterogenitet af hver ø celletype. Laser opsamling microdissection (LCM) teknik er blevet anvendt til direkte at opnå specifikke celletyper fra Holme9,10,11,12 , men falder kort af renhed af den målrettede celle Befolkning. For at overvinde disse begrænsninger er der anvendt fluorescens-aktiveret celle sortering (FACS) til at udvælge specifikke endokrine cellepopulationer, såsom α-og β-celler13,14,15,16 , 17 , 18. Desuden anvendte dorrell et al. en antistof-baseret FACS sortering tilgang til at klassificere β-celler i fire delpopulationer19. FACS-sorteret ø-celler kan også være belagt til RNA-sekvensering af enkeltceller; men de plade baserede metoder står over for udfordringer i skalerbarhed20,21,22.
For at generere høj kvalitet, storstilet transkriptomer data for hver endokrine celletype, vi anvendt mikrofluidisk teknologi til menneskelige ø-celler. Den mikrofluidisk platform genererer transkriptomer data fra et stort antal enkeltceller i en høj gennemløb, høj kvalitet, og skalerbar måde23,24,25,26,27. Ud over at afsløre molekylære karakteristika for en celletype, der er fanget i en stor mængde, muliggør yderst skalerbar mikrofluidisk-platform identifikation af sjældne celletyper, når der er nok celler til rådighed. Derfor, anvendelse af platformen til menneskelige pancreas Holme tilladt profilering af Ghrelin-secernerende ε-celler, en sjælden endokrine celletype med lidt kendt funktion på grund af sin knaphed28. I de seneste år er flere undersøgelser blevet offentliggjort af os og andre rapporterer store transkriptomer data af menneskelige Holme ved hjælp af teknologien29,30,31,32, 33. Dataene er offentligt tilgængelige og nyttige ressourcer for den ø samfund til at studere endokrine celle heterogenitet og dens konsekvenser i sygdomme.
Her beskriver vi en dråbe baseret mikrofluidic enkelt celle-RNA-sekvente Rings protokol, som er blevet brugt til at fremstille transkriptomdata på ca. 20.000 humane ø-celler, herunder α-, β-, δ-, PP-, ε-celler og en mindre del af ikke-endokrine celler 32. arbejdsprocessen starter med isolerede menneskelige Holme og skildrer trin af ø-celle dissociation, enkelt celle opsamling og dataanalyse. Protokollen kræver brug af frisk isolerede Holme og kan anvendes til Holme fra mennesker og andre arter, såsom gnavere. Ved hjælp af denne arbejdsgang, upartisk og omfattende ø-celle Atlas under baseline og andre betingelser kan bygges.
Protocol
Representative Results
Discussion
Single-celle teknologier udviklet i de seneste år giver en ny platform til at karakterisere celletyper og studere Molekylær heterogenitet i humane bugspytkirtel Holme. Vi vedtog en protokol af droplet-baseret mikrofluidisk enkelt celle isolation og dataanalyse for at studere menneskelige Holme. Vores protokol har med succes produceret RNA-sekvensering af data fra over 20.000 enkelt menneskelige ø-celler med relativt små variationer i sekvens kvalitet og batch effekter.
Især er to trin afg…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Ingen
Materials
| 30 µm Pre-Separation Filters | Miltenyi Biotec | 130-041-407 | Cell strainer |
| 8-chamber slides | Chemometec | 102673-680 | Dell counting assay slides |
| Bioanalyzer High Sensitivity DNA Kit | Agilent | 5067-4626 | for QC |
| Bovine Serum Albumin | Sigma-Aldrich | A9647 | Single cell media |
| Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns | 10X Genomics | 120237 | Single cell reagents |
| Chromium Single Cell A Chip Kit v2, 48 rx (6 chips) | 10X Genomics | 120236 | Microfluidic chips |
| CMRL-1066 | ThermoFisher | 11530-037 | Complete islet media |
| EB Buffer | Qiagen | 19086 | Elution buffer |
| Eppendorf twin-tec PCR plate, 96-well, blue, semi-skirted | VWR | 47744-112 | Emulsion plate |
| Fetal Bovine Serum | ThermoFisher | 16000-036 | Complete islet media |
| Human islets | Prodo Labs | HIR | Isolated human islets |
| L-Glutamine (200 mM) | ThermoFisher | 25030-081 | Complete islet media |
| Nextera DNA Library Preparation Kit (96 samples) | Illumina | FC-121-1031 | Library preparation reagents |
| NextSeq 500/550 High Output Kit v2.5 (75 cycles) | Illumina | FC-404-2005 | Sequencing |
| Penicillin-Streptomycin (10,000 U/mL) | ThermoFisher | 15140-122 | Complete islet media |
| Qubit High Sensitivity dsDNA Kit | Life Technologies | Q32854 | for QC |
| Solution 18 | Chemometec | 103011-420 | Cell counting assay reagent |
| SPRISelect Reagent | Fisher Scientific | B23318 | Purification beads |
| Tissue Culture Dishes (10 cm) | VWR | 10861-594 | for islet culture |
| TrypLE Express | Life Technologies | 12604-013 | Cell dissociation solution |
| Zymo DNA Clean & Concentrator-5, 50 reactions | VWR | 77001-152 | Library clean up columns |
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