Encellede transkriptom analyser af mus i bugspytkirtlen endokrine celler
Summary
Vi beskriver en metode til isolering af endokrine celler fra embryonale, neonatal og postnatal bugspytkirtel efterfulgt af encellede RNA sekventering. Denne metode giver mulighed for analyser af pancreas endokrine lineage udvikling, celle heterogenitet og transkriptom dynamik.
Abstract
Pancreas endokrine celler, der er grupperet i Holme, regulere blod glucose stabilitet og energimetabolisme. De forskellige celletyper i Holme, herunder insulin-secernerende β celler, er adskilt fra fælles endokrine stamfaderen under fosterstadiet. Umodne endokrine celler udvide via celleproliferation og modne i en lang postnatal udviklingsmæssige periode. Mekanismerne bag disse processer er imidlertid ikke klart defineret. Encellede RNA-sekventering er en lovende tilgang til karakterisering af forskellige cellepopulationer og spore celle lineage differentiering veje. Her, beskriver vi en metode til enkelt celle RNA-sekventering af isolerede pancreas β celler fra embryonale, neonatal og postnatal bugspytkirtel.
Introduction
Bugspytkirtlen er en vitale metaboliske organ i pattedyr. Bugspytkirtlen er sammensat af endokrine og eksokrine rum. Pancreas endokrine celler, herunder insulin-producerende β celler og glukagon-producerende α celler, klynge sammen i Holme i Langerhanske og regulere coordinately systemisk glukose homøostase. Dysfunktion af endokrine celler resultater i diabetes mellitus, som er blevet et stort folkesundhedsproblem på verdensplan.
Pancreas endokrine celler er afledt af Ngn3+ progenitorceller under embryogenese1. Senere, i den perinatale periode, de endokrine celler formere sig til form umodne Holme. Disse umodne celler vedbliver med at udvikle og efterhånden modne Holme, som bliver rigt vaskulariserede for at regulere blod glukose homøostase i voksne2.
Selv om en gruppe af transcriptional faktorer er blevet identificeret som regulerer β Celledifferentiering, er præcise modning pathway af β-celler stadig uklart. Β celle modning processen indebærer endvidere også reguleringen af celle nummer ekspansion3,4 og generation af cellulære heterogenitet5,6. Dog har reguleringsmekanismer af disse processer ikke været godt undersøgt.
Encellede RNA-sekventering er en kraftfuld tilgang, der kan profilere celle delpopulationer og spore celle lineage udviklingsmæssige veje7. Drage fordel af denne teknologi, nøglen hændelser, der opstår under pancreas islet udvikling kan være tydet på én celle niveau8. Blandt de encellede RNA-sekventering protokoller, Smart-seq2 giver mulighed for generation af fuld længde cDNA med forbedret følsomhed og nøjagtighed, og brugen af referencereagenser på lavere omkostninger9. Smart-seq2 tager ca. to dage til at konstruere en cDNA bibliotek for sekventering10.
Her foreslår vi en metode til isolering af fluorescens-mærket β celler fra bugspytkirtel af føtal til voksen Ins1-RFP Transgene mus11, ved hjælp af fluorescens-aktiveret celle sortering (FACS), og udførelsen af transkriptom analyserer på den encellede niveau, ved hjælp af Smart-seq2 teknologi (figur 1). Denne protokol kan udvides til at analysere transcriptomes af alle i bugspytkirtlen endokrine celletyper i aging, normale og patologiske stater.
Protocol
Representative Results
Discussion
I denne protokol demonstrerede vi en effektiv og nem at bruge metode til at studere encellede udtrykket profiler af pancreas β celler. Denne metode kan bruges til at isolere endokrine celler fra embryonale, neonatal og postnatal bugspytkirtel og udføre encellede transkriptom analyser.
Den mest kritiske trin er isolering af enkelt β celler i god stand. Fuldt perfunderet bugspytkirtel reagerer bedre på efterfølgende fordøjelsen. Utilstrækkelig perfusion, som normalt opstår i den dorsale …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker det nationale Center for Protein videnskaber, Beijing (Peking University) og Peking-Tsinghua Center for Life Science Computing Platform. Dette arbejde blev støttet af Ministeriet for videnskab og teknologi i Kina (2015CB942800), National Natural Science Foundation of China (31521004, 31471358 og 31522036) og støtte fra Peking-Tsinghua Center for Life Sciences til C.-R.X.
Materials
| Collagenase P | Roche | 11213873001 | |
| Trypsin-EDTA (0.25 %), phenol red | Thermo Fisher Scientific | 25200114 | |
| Fetal bovine serum (FBS) | Hyclone | SH30071.03 | |
| Dumont #4 Forceps | Roboz | RS-4904 | |
| Dumont #5 Forceps | Roboz | RS-5058 | |
| 30 G BD Needle 1/2" Length | BD | 305106 | |
| Stereo Microscope | Zeiss | Stemi DV4 | |
| Stereo Fluorescence microscope | Zeiss | Stereo Lumar V12 | |
| Centrifuge | Eppendorf | 5810R | |
| Centrifuge | Eppendorf | 5424R | |
| Polystyrene Round-Bottom Tube with Cell-Strainer Cap | BD-Falcon | 352235 | |
| 96-Well PCR Microplate | Axygen | PCR-96-C | |
| Silicone Sealing Mat | Axygen | AM-96-PCR-RD | |
| Thin Well PCR Tube | Extragene | P-02X8-CF | |
| Cell sorter | BD Biosciences | BD FACSAria | |
| Capillary pipette | Sutter | B100-58-10 | |
| RNaseZap | Ambion | AM9780 | |
| ERCC RNA Spike-In Mix | Life Technologies | 4456740 | |
| Distilled water | Gibco | 10977 | |
| Triton X-100 | Sigma-Aldrich | T9284 | |
| dNTP mix | New England Biolabs | N0447 | |
| Recombinant RNase Inhibitor | Takara | 2313 | |
| Superscript II reverse transcriptase | Invitrogen | 18064-014 | |
| First-strand buffer (5x) | Invitrogen | 18064-014 | |
| DTT | Invitrogen | 18064-014 | |
| Betaine | Sigma-Aldrich | 107-43-7 | |
| MgCl2 | Sigma-Aldrich | 7786-30-3 | |
| Nuclease-free water | Invitrogen | AM9932 | |
| KAPA HiFi HotStart ReadyMix (2x) | KAPA Biosystems | KK2601 | |
| VAHTS DNA Clean Beads XP beads | Vazyme | N411-03 | |
| Qubit dsDNA HS Assay Kit | Invitrogen | Q32854 | |
| AceQ qPCR SYBR Green Master Mix | Vazyme | Q121-02 | |
| TruePrep DNA Library Prep Kit V2 for Illumina | Vazyme | TD502 | Include 5x TTBL, 5x TTE, 5x TS, 5x TAB, TAE |
| TruePrep Index Kit V3 for Illumina | Vazyme | TD203 | Include 16 N6XX and 24 N8XX |
| High Sensitivity NGS Fragment Analysis Kit | Advanced Analytical Technologies | DNF-474 | |
| 1x HBSS without Ca2+ and Mg2+ | 138 mM NaCl; 5.34 mM KCl 4.17 mM NaHCO3; 0.34 mM Na2HPO4 0.44 mM KH2PO4 |
||
| Isolation buffer | 1 × HBSS containing 10 mM HEPES, 1 mM MgCl2, 5 mM Glucose, pH 7.4 | ||
| FACS buffer | 1 × HBSS containing 15 mM HEPES, 5.6 mM Glucose, 1% FBS, pH 7.4 | ||
| NaCl | Sigma-Aldrich | S5886 | |
| KCl | Sigma-Aldrich | P9541 | |
| NaHCO3 | Sigma-Aldrich | S6297 | |
| Na2HPO4 | Sigma-Aldrich | S5136 | |
| KH2PO4 | Sigma-Aldrich | P5655 | |
| D-(+)-Glucose | Sigma-Aldrich | G5767 | |
| HEPES | Sigma-Aldrich | H4034 | |
| MgCl2 | Sigma-Aldrich | M2393 | |
| Oligo-dT30VN primer | 5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3' | ||
| TSO | 5'-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3' | ||
| ISPCR primers | 5'-AAGCAGTGGTATCAACGCAGAGT-3' | ||
| Gapdh Forward primer | 5'-ATGGTGAAGGTCGGTGTGAAC-3' | ||
| Gapdh Reverse primer | 5'-GCCTTGACTGTGCCGTTGAAT-3' | ||
| Ins2 Forward primer | 5'-TGGCTTCTTCTACACACCCA-3' | ||
| Ins2 Reverse primer | 5'-TCTAGTTGCAGTAGTTCTCCA-3' |
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