Genanvendelig enkeltcelle til iterative epigenomiske analyser
Summary
Denne protokol beskriver en enkeltcellemetode til iterative epigenomiske analyser ved hjælp af en genanvendelig enkeltcelle. Den genanvendelige enkeltcelle muliggør analyser af flere epigenetiske mærker i samme enkeltcelle og statistisk validering af resultaterne.
Abstract
Nuværende enkeltcelle epigenomanalyser er designet til engangsbrug. Cellen kasseres efter en enkelt brug, hvilket forhindrer analyse af flere epigenetiske mærker i en enkelt celle og kræver data fra andre celler for at skelne signal fra eksperimentel baggrundsstøj i en enkelt celle. Dette papir beskriver en metode til at genbruge den samme enkelte celle til iterative epigenomiske analyser.
I denne eksperimentelle metode er cellulære proteiner først forankret til en polyacrylamidpolymer i stedet for at krydsbinde dem til protein og DNA, hvilket lindrer strukturel bias. Dette kritiske trin tillader gentagne eksperimenter med den samme enkeltcelle. Derefter udglødes en tilfældig primer med en stilladssekvens til nærhedsligering til det genomiske DNA, og den genomiske sekvens tilsættes primeren i forlængelse heraf under anvendelse af en DNA-polymerase. Derefter er et antistof mod en epigenetisk markør og kontrol IgG, hver mærket med forskellige DNA-sonder, bundet til de respektive mål i den samme enkeltcelle.
Nærhedsligering induceres mellem den tilfældige primer og antistoffet ved at tilføje et forbindelses-DNA med komplementære sekvenser til stilladssekvensen af den tilfældige primer og antistof-DNA-sonden. Denne tilgang integrerer antistofinformation og nærliggende genomsekvenser i et enkelt DNA-produkt af nærhedsligering. Ved at muliggøre gentagne eksperimenter med den samme enkeltcelle tillader denne metode en stigning i datatæthed fra en sjælden celle og statistisk analyse ved kun at bruge IgG- og antistofdata fra den samme celle. De genanvendelige enkeltceller fremstillet ved denne metode kan opbevares i mindst et par måneder og genbruges senere for at udvide epigenetisk karakterisering og øge datatætheden. Denne metode giver fleksibilitet til forskere og deres projekter.
Introduction
Enkeltcelleteknologi går ind i æraen med enkeltcelle multiomics, som integrerer individuelle enkeltcelle omics-teknologier1. For nylig er enkeltcelletranskriptomik blevet kombineret med metoder til påvisning af kromatintilgængelighed (scNMT-seq2 og SHARE-seq3) eller histonmodifikationer (Paired-seq4 og Paired-Tag5). For nylig blev enkeltcelletranskriptomik og proteomik integreret med kromatintilgængelighed (DOGMA-seq6). Disse metoder bruger transposasebaseret tagging til påvisning af kromatintilgængelighed eller histonmodifikationer.
Transposasebaserede tilgange spalter genomisk DNA og tilføjer en DNA-stregkode i slutningen af det genomiske DNA-fragment. Hvert spaltet genomisk fragment kan kun acceptere op til to DNA-stregkoder (= et epigenetisk mærke pr. spaltningssted), og det genomiske DNA på spaltningsstedet går tabt. Derfor har spaltningsbaserede tilgange en afvejning mellem antallet af testede epigenetiske mærker og signaltætheden. Dette hæmmer analysen af flere epigenetiske mærker i samme enkeltcelle. En enkeltcelle epigenomisk metode, der ikke spalter det genomiske DNA, blev udviklet for at overvinde dette problem 7,8.
Ud over det spaltningsafledte problem, der er nævnt ovenfor, har transposasebaserede tilgange andre begrænsninger. I enkeltcelle epigenomanalyse er det afgørende at kende placeringen af histoner og DNA-associerede proteiner på genomet. I nuværende tilgange opnås dette ved at bruge ikke-fikserede enkeltceller og tilbageholdelse af protein-DNA og protein-proteininteraktioner. Dette genererer imidlertid stærk bias til tilgængelige kromatinregioner, selv i analysen af histonmodifikationer 9. Placeringen af histoner og genomassocierede proteiner på genomet kan bevares uden tværbinding af protein-DNA og proteinprotein ved hjælp af et polyacrylamidstillads 7,8. Denne tilgang reducerer den strukturelle bias, der observeres i nuværende tilgange, der afhænger af protein-DNA og protein-proteininteraktioner.
Transposasebaserede tilgange kan kun erhverve signaler én gang fra en enkelt celle. Derfor er det vanskeligt at afgrænse det komplette epigenom af en enkelt celle på grund af signalernes drop-off. Genanvendelige enkeltceller er udviklet til at overvinde de nuværende begrænsninger ved at tillade iterativ epigenomisk analyse i den samme enkeltcelle.
Protocol
Representative Results
Discussion
Denne artikel beskriver den trinvise protokol for den nyligt rapporterede enkeltcellede multiepigenomiske analyse ved hjælp af genanvendelige enkeltceller7. I de efterfølgende afsnit diskuterer vi kritiske punkter og understreger potentielle begrænsninger i protokollen.
Et af de kritiske punkter i hele protokollen (fra trin 7.2-13) er at undgå DNase-kontaminering. En enkelt celle har kun to kopier af genomisk DNA. Derfor reducerer skadeligt genomisk DNA signalantal…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker Dr. David Sanchez-Martin og Christopher B. Buck for kommentarer i projektets konceptualiseringsfase. Vi takker også Genomics Core, Center for Cancer Research, National Cancer Institute, National Institutes of Health for hjælp i indledende eksperimenter og Collaborative Bioinformatics Resource, CCR, NCI, NIH for rådgivning i beregningsanalyse. Vi takker Anna Word for at hjælpe med optimeringen af DNA-polymeraser, der anvendes i metoden. Dette arbejde udnyttede beregningsressourcerne i NIHHPC Biowulf-klyngen (http://hpc.nih.gov). Dette projekt støttes af det intramurale program fra Center for Kræftforskning, National Cancer Institute, National Institutes of Health, NCI Director’s Innovation Award (# 397172) og føderale midler fra National Cancer Institute under kontrakt nr. HHSN261200800001E. Vi takker Dr. Tom Misteli, Carol Thiele, Douglas R. Lowy og alle medlemmer af Laboratory of Cellular Oncology for produktive kommentarer.
Materials
| 10x CutSmart buffer | New England BioLabs | B6004 | 10x Digestion buffer |
| 200 proof ethanol | Warner-Graham Company | 200 proof | Ethanol |
| 5-Hydroxymethylcytosine (5-hmC) Monoclonal Antibody [HMC/4D9] | Epigentek | A-1018-100 | Anti-5hmC |
| Acridine Orange/Propidium Iodide Stain | Logos Biosystems | F23001 | Cell counter |
| Acrylamide solution, 40% in H2O, for molecular biology | MilliporeSigma | 01697-500ML | 40% acrylamide solution |
| All-in-One Fluorescence Microscope BZ-X710 | Keyence | BZ-X710 | Scanning microscope |
| Amicon Ultra-0.5 Centrifugal Filter Unit | MilliporeSigma | UFC510024 | Ultrafiltration cassette |
| Ammonium persulfate for molecular biology | MilliporeSigma | A3678-100G | Ammonium persulfate powder |
| Anhydrous DMF | Vector laboratories | S-4001-005 | Anhydrous N,N-dimethylformamide (DMF) |
| Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] | Abcam | ab5408 | Anti-Pol II |
| Anti-TRAP220/MED1 (phospho T1457) antibody | Abcam | ab60950 | Anti-Med1 |
| BciVI | New England BioLabs | R0596L | BciVI |
| Bovine Serum Albumin solution, 20 mg/mL in H2O, low bioburden, protease-free, for molecular biology | MilliporeSigma | B8667-5ML | 20% BSA (Table 7) |
| Bst DNA Polymerase, Large Fragment | New England BioLabs | M0275L | Bst DNA polymerase |
| BT10 Series 10 µl Barrier Tip | NEPTUNE | BT10 | P10 low-retention tip |
| CellCelector | Automated Lab Solutions | N/A | Automated single cell picking robot |
| CellCelector 4 nl nanowell plates for single cell cloning, Plate S200-100 100K, 24 well,ULA | Automated Lab Solutions | CC0079 | 4 nL nanowell plate |
| Chloroform | MilliporeSigma | Chloroform | |
| Corning Costar 96-Well, Cell Culture-Treated, Flat-Bottom Microplate | Corning | 3596 | Flat-bottom 96-well plates |
| Deep Vent (exo-) DNA Polymerase | New England BioLabs | M0259L | Exo– DNA polymerase |
| DNA LoBind Tubes, 0.5 mL | Eppendorf | 30108035 | 0.5 mL DNA low-binding tube |
| DNA Oligo, 1st random primer | Integrated DNA Technologies | N/A, see Table 3 | 1st random primer |
| DNA Oligo, 2nd random primer Cell#01 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#02 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#03 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#04 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#05 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#06 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#07 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#08 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#09 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#10 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#11 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#12 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd synthesis primer | Integrated DNA Technologies | N/A, see Table 3 | 2nd synthesis primer |
| DNA Oligo, Ligation Adaptor | Integrated DNA Technologies | N/A, see Table 3 | Ligation Adaptor |
| DNA Oligo, Reverse Transcription primer | Integrated DNA Technologies | N/A, see Table 3 | Reverse Transcription primer |
| DNase I (RNase-free) | New England BioLabs | M0303L | DNase I (RNase-free, 4 U). |
| DNase I Reaction Buffer | New England BioLabs | B0303S | 10x DNase I buffer (NEB) |
| dNTP Mix (10 mM each) | Thermo Fisher | R0192 | 10 mM dNTPs |
| Fetal Bovine Serum, USA origin, Heat-inactivated | MilliporeSigma | F4135-500ML | Fetal bovine serum |
| HiScribe T7 High Yield RNA Synthesis Kit | New England BioLabs | E2040S | In-vitro-transcription master mix |
| Histone H3K27ac antibody | Active motif | 39133 | Anti-H3K27ac |
| Histone H3K27me3 antibody | Active motif | 39155 | Anti-H3K27me3 |
| IgG from rabbit serum | Millipore Sigma | I5006-10MG | Control IgG |
| Iron oxide(II,III) magnetic nanopowder, 30 nm avg. part. size (TEM), NHS ester functionalized | MilliporeSigma | 747467-1G | NHS ester functionalized 30 nm iron oxide powder |
| K-562 | American Type Culture Collection (ATCC) | CCL-243 | cells |
| Linear Acrylamide (5 mg/mL) | Thermo Fisher | AM9520 | Linear Acrylamide |
| LUNA-FL Dual Fluorescence Cell Counter | Logos Biosystems | L20001 | Cell counter |
| LUNA Cell Counting Slides, 50 Slides | Logos Biosystems | L12001 | Cell counter |
| Mineral oil, BioReagent, for molecular biology, light oil | MilliporeSigma | M5904-500ML | Mineral oil |
| N,N,N′,N′-Tetramethylethylenediamine for molecular biology | MilliporeSigma | T7024-100ML | N,N,N′,N′-Tetramethylethylenediamine |
| NaCl (5 M), RNase-free | Thermo Fisher | AM9760G | 5M NaCl |
| NanoDrop Lite | Thermo Fisher | 2516 | Microvolume spectrophotometer |
| NEST 2 mL 96-Well Deep Well Plate, V Bottom | Opentrons | N/A | 2 mL deep well 96-well plate |
| Non-skirted 96-well PCR plate | Genesee Scientific | 27-405 | 96-well PCR plate |
| NuSive GTG Agarose | Lonza | 50081 | Agarose |
| OmniPur Acrylamide: Bis-acrylamide 19:1, 40% Solution | MilliporeSigma | 1300-500ML | 40%Acrylamide/Bis-acrylamide |
| OT-2 lab robot | Opentrons | OT2 | Automated liquid handling robot |
| Paraformaldehyde, EM Grade, Purified, 20% Aqueous Solution | Electron Microscopy Sciences | 15713 | 20% Pararmaldehyde |
| PBS (10x), pH 7.4 | Thermo Fisher | 70011044 | 10x PBS |
| PIPETMAN Classic P1000 | GILSON | F123602 | A P1000 pipette |
| Protein LoBind Tubes, 1.5 mL | Eppendorf | 925000090 | 1.5 mL Protein low-binding tube |
| QIAgen Gel Extraction kit | Qiagen | 28706 | A P1000 pipette |
| Quant-iT PicoGreen dsDNA Assay | Thermo Fisher | P11495 | dsDNA specific intercalator dye |
| Quick Ligation kit | New England BioLabs | M2200L | T4 DNA ligase (NEB) |
| RNaseOUT Recombinant Ribonuclease Inhibitor | Thermo Fisher | 10777019 | RNAse inhibitor |
| S-4FB Crosslinker (DMF-soluble) | Vector laboratories | S-1004-105 | Succinimidyl 4-formylbenzoate (S-4FB) |
| S-HyNic | Vector laboratories | S-1002-105 | Succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic) |
| Sodium Acetate, 3 M, pH 5.2, Molecular Biology Grade | MilliporeSigma | 567422-100ML | 3M Sodium acetate (pH 5.2) |
| Sodium bicarbonate, 1M buffer soln., pH 8.5 | Alfa Aesar | J60408 | 1M sodium bicarbonate buffer, pH 8.5 |
| Sodium phosphate dibasic for molecular biology | MilliporeSigma | S3264-250G | Na2HPO4 |
| Sodium phosphate monobasic for molecular biology | MilliporeSigma | S3139-250G | NaH2PO4 |
| SuperScript IV reverse transcriptase | Thermo Fisher | 18090050 | Reverse transcriptase |
| SYBR Gold Nucleic Acid Gel Stain (10,000x Concentrate in DMSO) | Thermo Fisher | S11494 | An intercalator dye for DNA |
| T4 DNA Ligase Reaction Buffer | New England BioLabs | B0202S | 10x T4 DNA ligase reaction buffer |
| ThermoPol Reaction Buffer Pack | New England BioLabs | B9004S | 10x TPM-T buffer (Tris-HCl/Pottasium chloride/Magnesium sulfate/Triton X-100) |
| TRIzol LS reagent | Thermo Fisher | 10296-028 | Guanidinium thiocyanate-phenol-chloroform extraction |
| TruSeq Nano DNA library prep kit | Illumina | 20015965 | A DNA library preparation kit (see also the manufacturer's instruction) |
| Ultramer DNA Oligo, Anti-5hmC_Ab#005 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
| Ultramer DNA Oligo, Anti-H3K27ac_Ab#002 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
| Ultramer DNA Oligo, Anti-H3K27me3_Ab#003 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
| Ultramer DNA Oligo, Anti-Med1_Ab#004 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
| Ultramer DNA Oligo, Anti-Pol II_Ab#006 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
| Ultramer DNA Oligo, Control IgG_Ab#001 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for control IgG |
| UltraPure 0.5 M EDTA, pH 8.0 | Thermo Fisher | 15575020 | 0.5M EDTA, pH 8.0 |
| UltraPure DNase/RNase-Free Distilled Water | Thermo Fisher | 10977023 | Ultrapure water |
| Zeba Splin Desalting Columns, 7K MWCO, 0.5 mL | Thermo Fisher | 89882 | Desalting column |
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