Kortlægning Genome-wide tilgængelige kromatin i primære humane T lymfocytter af ATAC-Seq
Summary
Assay for Transposase-tilgængelige kromatin kombineret med høj overførselshastighed sekventering (ATAC-FF.) er en genome-wide metode til at afdække tilgængelige kromatin. Dette er en trinvis ATAC-seq-protokollen fra molekylære til den endelige beregningsmæssige analyse, optimeret til humane lymfocytter (Th1/Th2). Denne protokol kan vedtages af forskere uden forudgående erfaring i næste generations sekventering metoder.
Abstract
Assay for Transposase-tilgængelige kromatin med høj overførselshastighed sekventering (ATAC-FF.) er en metode, der anvendes til identifikation af åbne (tilgængelige) regioner af kromatin. Disse områder repræsenterer regulerende DNA elementer (f.eks.promotorer, smagsforstærkere, locus kontrol regioner, isolatorer) til hvilke transskription faktorer binde. Kortlægning af de tilgængelige kromatin landskab er en kraftfuld tilgang til afsløring aktive regulerende elementer på tværs af genomet. Disse oplysninger tjener som en fordomsfri tilgang for at opdage netværket af relevante transkriptionsfaktorer og mekanismer af kromatin struktur, der styrer gen expression programmer. ATAC-seq er en robust og følsom alternativ til DNase jeg overfølsomhed analyse kombineret med næste generation sequencing (DNase-seq) og formaldehyd-assisteret isolation af regulerende elementer (FAIRE-seq) for genome-wide analyse af kromatin tilgængelighed og til sekvensering af micrococcal nukleasen-følsomme steder (MNase-seq) til at bestemme nucleosome placering. Vi præsenterer en detaljeret ATAC-seq protokol optimeret nemlig menneskelige primære immun celler dvs CD4 + lymfocytter (T hjælper 1 (Th1) og Th2 celler). Denne omfattende protokol begynder med celle høst, så beskriver den molekylære procedure af kromatin tagmentation, prøveforberedelse til næste generations sekvensering, og også omfatter metoder og overvejelser i forbindelse med de beregningsmæssige analyser bruges til at fortolke resultaterne. Desuden, for at spare tid og penge, vi indført kvalitetskontrolforanstaltninger til at vurdere ATAC-seq biblioteket før sekvensering. Vigtigere, tillader de principper, der er præsenteret i denne protokol dets tilpasning til andre menneskers immunforsvar og ikke-immune primærelementer og cellelinjer. Disse retningslinjer vil også være nyttigt for laboratorier, der ikke er dygtige med næste generations sekventering metoder.
Introduction
ATAC-seq1,2 er en robust metode, der muliggør identifikation af regulerende3 åbne kromatin regioner og nucleosome placering. Disse oplysninger anvendes til at udlede placering, identitet og aktivitet af transkriptionsfaktorer. Metodens følsomhed for måling af de kvantitative variationer i kromatin struktur giver mulighed for undersøgelse af kromatin faktorer, herunder kromatin remodelers samt modificeringer og RNA polymerase II1transcriptional aktivitet aktivitet. Dermed giver ATAC-seq en kraftfuld og fordomsfri tilgang til decifrere mekanismer, der styrer transkriptionel regulering i enhver celletype af interesse. Vi beskriver tilpasningen af ATAC-seq til primære menneskelige Th1 og Th2 celler.
I ATAC-FF., hyperaktiv Tn5 transposase fyldt med adaptere til næste generation sequencing (NGS) par DNA opsplitning med mærkning af DNA med adaptere (dvs., at “tagmentation”)1. Efter PCR-amplifikation er de resulterende DNA biblioteker klar til næste generation sequencing (figur 1). Den privilegerede tagmentation af tilgængelige kromatin registreres af analysen af lokale berigelse af ATAC-seq sekventering læser.
Kort eksperimentel procedure og krav om mindre råvaren, i forhold til andre metoder til måling af kromatin tilgængelighed og nucleosomal positionering som DNase-seq4, FAIRE-seq5og MNase-seq6, har fremmet brugen af ATAC-seq i flere biologiske systemer, herunder menneskelige primærelementer1,7 kliniske prøver8, samt og encellede organismer9, planter10, bananfluer11 , og forskellige pattedyr12.
Identiteten af transskription faktorer, der er bundet til tilgængelige loci kan blive afsløret ved at analysere berigelsen af deres bindende sekvens motiver eller kombinere ATAC-seq med kromatin immunoprecipitation (ChIP) efterfulgt af høj overførselshastighed DNA-sekventering ( ChIP-seq). Denne tilgang aktiveret identifikation af afstamning-specifikke transkriptionsfaktorer vigtigt for bloddannelsen i mus13. ATAC-seq saglig og globale natur giver mulighed for at studere RIBOREGULATION i organismer som reagenser som antistoffer for ChIP analyse ikke er tilgængelige. For eksempel evolutionære variationer i cis-regulerende regioner er blevet identificeret ved at studere kraniel neurale crest celler fra mennesker og chimpanser14, udviklingsmæssige variationer i regulerende elementer under tidlige mus embryogenese15, ændringer i den lovgivningslandskab i løbet af en livscyklus af encellede C. owzarzaki9, og udviklingen af initiativtagerne og smagsforstærkere i hele 20 pattedyr arter12.
ATAC-seq har også været medvirkende til at måle kromatin tilgængelighed i enkelt celler, således afslørende variation inden for cellepopulationer, som normalt undviger genome-wide undersøgelser7,16. Derudover kan ATAC-seq bruges til at studere forandringer, der sker i DNA regulerende regioner i sygdomstilstande, hvor prøverne er sjældne. For eksempel, ATAC-seq kan bruges til at studere ændringer på den reguleringsmæssige landskab under udbruddet af akut myeloid leukæmi (AML)17 eller Ras-drevet oncogenesis11.
Protocol
Representative Results
Discussion
ATAC-seq protokollen beskrevet her har været med succes ansat til analyse af tilgængelige kromatin i primærelementer (menneskelige Th1, Th2 celler og B-celler) samt kulturperler cellelinjer (MCF10A humane brystkræftceller og U261 glioblastom celler). Anvende ATAC-seq til andre celletyper kan kræve nogle protokol optimering, især i trinnet lysering. Hvis koncentrationen af nonionisk detergent er for højt, kan der være en højere procentdel af mitokondriel DNA forurening. Dette kan reduceres ved at mindske koncentr…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde støttes af Israel Science Foundation (grant 748/14), Marie Curie-Integration yde (CIG) – RP7-folk-20013-CIG-618763 og CORE Program af planlægning og budgettering udvalg og The Israel Science Foundation give nr. 41/11.
Materials
| 50 mL tubes | Lumitron | LUM-CFT011500-P | Can be from other vendors. |
| Microtubes | Axygen Inc | MCT-175-C | Can be from other vendors. |
| 25 mL serological pipettes | Corning Costar | 4489 | Can be from other vendors. |
| Tissue culture flask | Lumitron | LUM-TCF-012-250-P | Can be from other vendors. |
| Countes Automated Cell Counter | Invitrogen | C10227 | |
| NucleoSpin Tissue | MACHEREY-NAGEL | 740952.5 | |
| Peripheral blood mononuclear cells (PBMC) | ATCC | PCS800011 | Can be from other vendors. |
| RPMI 1640 Medium | Biological Industries | 01-103-1A | Can be from other vendors. |
| L-Glutamine Solution (200 mM) | Biological Industries | 03-020-1B | Can be from other vendors. |
| Penicillin-Streptomycin | Biological Industries | 03-031-1B | Can be from other vendors. |
| Fetal Bovine Serum (FBS), Heat Inactivated, European Grade | Biological Industries | 04-127-1 | Can be from other vendors. |
| MACS CD4 microbeads, human | Miltenyi Biotec | 130-045-101 | |
| MACS MS columns | Miltenyi Biotec | 130-042-201 | |
| Anti-Human CD4 FITC | Biogems | 06121-50 | |
| Mouse IgG1 Isotype Control FITC | Biogems | 44212-50 | |
| Anti-Human CD3 (OKT3) | Tonbo biosciences | 40-0037 | |
| Anti-Human CD28 SAFIRE Purified | Biogems | 10311-25 | |
| Recombinant Human IL2 | Peprotech | 200-02 | |
| Recombinant Human IL4 | Peprotech | 200-04 | |
| Recombinant Human IL12 p70 | Peprotech | 200-12 | |
| In Vivo Ready Anti-Human IL-4 (MP4-25D2) | Tonbo | 40-7048 | |
| LEAF Purified anti-human IFN-γ | BioLegend | 506513 | |
| NaCl, analytical grade | Carlo Erba | 479687 | Can be from other vendors. |
| Magnesium chloride, Hexahydrate, molecular biology grade | Calbiochem | 442611 | Can be from other vendors. |
| EDTA | MP Biomedicals | 800682 | Can be from other vendors. |
| Tris, ultra pure, 99.9% pure | MP Biomedicals | 819620 | Can be from other vendors. |
| NP-40 alternative (Nonylphenyl Polyethylene Glycol) | Calbiochem | 492016 | Can be from other vendors. |
| Protease Inhibitors | Sigma | P2714 | this protease inhibitor coctail is a powder. To make 100 x solution dilute in 1 mL of molecular-biology grade water. |
| Magnetic solid phase reverse immobilization beads: AMPure XP beads | Beckman | 63881 | |
| PCR purification kit | HyLabs | EX-GP200 | Can be from other vendors. |
| Nextera DNA Library Preparation Kit (TDE1 transposase and TD buffer) | Illumina | FC-121-1030 | |
| NEBNext High-Fidelity 2 x PCR Master Mix | New England BioLabs | M0541 | |
| NEBNext Q5 Hot Start HiFi PCR Master Mix | New England BioLabs | M0543 | |
| SYBR Green I | Invitrogen | S7585 | |
| CFX Connect Real-Time PCR Detection System | Bio-rad | 185-5200 | Can be from other vendors. |
| CFX Manager Software | Bio-rad | 1845000 | |
| master mix for qPCR: iTaq Universal SYBR Green Supermix | Bio-rad | 172-5124 | Can be from other vendors. |
| Qubit fluorometer 2.0 | Invitrogen | Q32866 | |
| Qubit dsDNA HS Assay Kit | Invitrogen | Q32854 | |
| Magnet for eppendorf tubes | Invitrogen | 12321D | Can be from other vendors. |
| Swing bucket cooling centrifuge with the buckets for 15 mL falcon tubes and eppendorf tubes | Thermo Scientific | 75004527 | Could be from other vendors. It is important that it has buckets for eppendorf tubes. |
| Thermo-shaker | MRC | Can be from other vendors. | |
| High Sensitivity D1000 ScreenTape | Agilent Technologies | 5067-5584 | |
| High Sensitivity D1000 Reagents | Agilent Technologies | 5067-5585 | |
| 4200 TapeStation system | Agilent Technologies | G2991AA | Tape-based platform for electrophoresis |
| High Sensitivity DNA kit | Agilent Technologies | 5067-4626 | Reagent for high-sensitivity TapeStation analysis |
| Primer name and sequence | Company | ||
| Ad1_noMX: 5'-AATGATACGGCGACCACCGAGA TCTACACTCGTCGGCAGCGTC AGATGTG-3' |
IDT | Ad1-noMx: 5'-P5 sequence-transposase sequence-3' | |
| Ad2.1_TAAGGCGA: 5'-CAAGCAGAAGACGGCATACGAG AT[TCGCCTTA]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.1_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.2_CGTACTAG: 5'-CAAGCAGAAGACGGCATACGAG AT[CTAGTACG]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.2_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.3_AGGCAGAA: 5'-CAAGCAGAAGACGGCATACGA GAT[TTCTGCCT]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.3_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.4_TCCTGAGC: 5'-CAAGCAGAAGACGGCATACGAG AT[GCTCAGGA]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.4_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.5_GGACTCCT: 5'-CAAGCAGAAGACGGCATACGA GAT[AGGAGTCC]GTCTCGTGGG CTCGGAGATGT-3' |
IDT | Ad2.5_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.6_TAGGCATG: 5'-CAAGCAGAAGACGGCATACGA GAT[CATGCCTA]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.6_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.7_CTCTCTAC: 5'-CAAGCAGAAGACGGCATACGA GAT[GTAGAGAG]GTCTCGTGGG CTCGGAGATGT-3' |
IDT | Ad2.7_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.8_CAGAGAGG: 5'-CAAGCAGAAGACGGCATACGA GAT[CCTCTCTG]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.8_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.9_GCTACGCT: 5'-CAAGCAGAAGACGGCATACGA GAT[AGCGTAGC]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.9_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.10_CGAGGCTG: 5'-CAAGCAGAAGACGGCATACG AGAT[CAGCCTCG]GTCTCGTGG GCTCGGAGATGT-3' |
IDT | Ad2.10_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.11_AAGAGGCA: 5'-CAAGCAGAAGACGGCATACG AGAT[TGCCTCTT]GTCTCGTGGG CTCGGAGATGT-3' |
IDT | Ad2.11_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.12_GTAGAGGA: 5'-CAAGCAGAAGACGGCATACG AGAT[TCCTCTAC]GTCTCGTGGG CTCGGAGATGT-3' |
IDT | Ad2.12_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.13_GTCGTGAT: 5'-CAAGCAGAAGACGGCATACGA GAT[ATCACGAC]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.13_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.14_ACCACTGT: 5'- CAAGCAGAAGACGGCATACGA GAT[ACAGTGGT]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.14_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.15_TGGATCTG: 5'- CAAGCAGAAGACGGCATACGA GAT[CAGATCCA]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.15_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.16_CCGTTTGT: 5'- CAAGCAGAAGACGGCATACGA GAT[ACAAACGG]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.16_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.17_TGCTGGGT: 5'- CAAGCAGAAGACGGCATACGA GAT[ACCCAGCA]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.17_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.18_GAGGGGTT: 5'-CAAGCAGAAGACGGCATACGA GAT[AACCCCTC]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.18_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.19_AGGTTGGG: 5'-CAAGCAGAAGACGGCATACGA GAT[CCCAACCT]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.19_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.20_GTGTGGTG: 5'-CAAGCAGAAGACGGCATACGA GAT[CACCACAC]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.20_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.21_TGGGTTTC: 5'-CAAGCAGAAGACGGCATACGA GAT[GAAACCCA]GTCTCGTGGGC TCGGAGATGT-3' |
IDT | Ad2.21_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.22_TGGTCACA: 5'- CAAGCAGAAGACGGCATACGA GAT[TGTGACCA]GTCTCGTGGGCT CGGAGATGT-3' |
IDT | Ad2.22_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.23_TTGACCCT: 5'-CAAGCAGAAGACGGCATACGA GAT[AGGGTCAA]GTCTCGTGGGCT CGGAGATGT-3' |
IDT | Ad2.23_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| Ad2.24_CCACTCCT: 5'-CAAGCAGAAGACGGCATACGA GAT[AGGAGTGG]GTCTCGTGGGCT CGGAGATGT-3' |
IDT | Ad2.24_expected index sequence read: 5'-P7 sequence-[index sequence]-transposase sequence-3' | |
| F1: 5'-CCTTTTTATTTGCCCATACACTC-3' | IDT | ||
| R1: 5'-CCCAGATAGAAAGTTGGAGAGG-3' | IDT | ||
| F2: 5'-TTGAGGGATGCCATAACAGTC-3' | IDT | ||
| R2: 5'-CTGCTGAACAACATCCTTCAC-3' | IDT | ||
| F3: 5'-GGTTTGCAGGTTGCGTTG-3' | IDT | ||
| R3: 5'-AGAGGAATCTGGGAGTGACG-3' | IDT | ||
| F4: 5'-TGCTCATTCCGTTTCCCTAC-3' | IDT | ||
| R4: 5'-AGCCGGAAAGAAAGTTCCTG-3' | IDT |
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