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Ukusebenzisana kwe-Chromatin okusekelwe ku-Hi-C
I-Hi-C iyindlela eyenzelwe ukubamba ukwakheka kwezakhi zofuzo ngokuhlanganisa ukusebenzisana okusekelwe ekuhloleni ukusondelana kanye nokulandelana okuphezulu kokuphuma. Le ndlela isekelwe ekuxhumeni kwe-chromatin ne-formaldehyde, kulandelwe ukugaya kanye nokuhlanganiswa kabusha ngendlela yokuthi izingcezu ezixhumene ndawonye kuphela ezizokwakha imikhiqizo yokuhlanganiswa. Ngokulandelanisa le mikhiqizo yokuhlanganiswa, kungenzeka ukutadisha inhlangano ye-3D ye-genome. I-Hi-C ivumela ukutadisha ukusatshalaliswa kwezingxenye ze-genome ezipakishwe kancane (ama-compartments e-A, i-euchromatin) futhi okungenzeka ukuthi zisebenza ngokubhala, kanye nezifunda ezipakishwe kakhulu (ama-compartments e-B, i-Heterochromatin). I-Hi-C ingasetshenziswa futhi ukukhomba ama-Topologically Associated Domains (ama-TAD), izifunda ze-genome ezinezakhiwo ezigoqiwe futhi cishe zinezindlela ezifanayo zokuveza, kanye nokuhlonza ama-chromatin loops, izifunda ze-DNA ezihlanganiswe ndawonye ngamaprotheni futhi ezivame ukucebiswa ezintweni ezilawulayo. Insizakalo yokulandelelana kwe-BMKGene ye-Hi-C inika abacwaningi amandla okuhlola ubukhulu bendawo be-genomics, ivula izindlela ezintsha zokuqonda ukulawulwa kwe-genome kanye nemiphumela yayo empilweni nasezifweni.
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Ukulandelana Kwe-Chromatin Immunoprecipitation (ChIP-seq)
I-Chromatin Immunoprecipitation (CHIP) iyindlela esebenzisa ama-antibodies ukuze ikhethe ngokukhethekile amaprotheni ahlanganisa i-DNA kanye nezinhloso zawo ze-genomics. Ukuhlanganiswa kwayo ne-NGS kwenza kube lula ukuqoshwa kwe-genome yonke kwezinhloso ze-DNA ezihlobene nokuguqulwa kwe-histone, izici zokubhala, kanye namanye amaprotheni ahlanganisa i-DNA. Le ndlela enamandla ivumela ukuqhathaniswa kwezindawo zokubopha kuzo zonke izinhlobo zamaseli, izicubu, noma izimo. Ukusetshenziswa kwe-ChIP-Seq kuqala ekutadisheni ukulawulwa kokubhala kanye nezindlela zokuthuthukiswa kuya ekuchazeni izindlela zesifo, okwenza kube ithuluzi elibalulekile lokuqonda izindawo zokulawulwa kokuzalwa kanye nokuthuthukisa ukuqonda kokwelapha.
Ipulatifomu: Illumina NovaSeq
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Ukulandelana kwe-bisulfite ye-genome ephelele (WGBS)
Le ndlela, esekelwe ekwelashweni kwe-bisulfite kwe-DNA ukuze kuguqulwe ama-cytosine angena-methylated abe yi-uracil (C kuya ku-U), kuyilapho ishiya ama-cytosine angena-methylated engashintshiwe, iyindlela ejwayelekile yokuhlola ngokujulile i-methylation ye-DNA, ikakhulukazi indawo yesihlanu ku-cytosine (5-mC), umlawuli obalulekile wokubonakaliswa kwezakhi zofuzo kanye nomsebenzi wamaseli.
Le ndlela inikeza isisombululo sesisekelo esisodwa, okuvumela abacwaningi ukuthi baphenye ngokuphelele i-methylome futhi bathole amaphethini e-methylation angavamile ngaphakathi kwamasampula. Ngokusebenzisa i-WGBS, ososayensi bangathola ukuqonda okungenakuqhathaniswa mayelana nezindawo ze-methylation ezibanzi ze-genome, okuhlinzeka ngokuqonda okuningiliziwe kwezindlela ze-epigenetic ezisekela izinqubo nezifo ezahlukahlukene zezinto eziphilayo.
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Ukuhlolwa kwe-Chromatin efinyeleleka kalula ye-Transposase ene-High Throughput Sequencing (ATAC-seq)
I-ATAC-seq iyindlela yokulandelana kwe-high-throughput esetshenziselwa ukuhlaziywa kokufinyeleleka kwe-chromatin ku-genome. Ukusetshenziswa kwayo kunikeza ukuqonda okujulile kwezindlela eziyinkimbinkimbi zokulawula i-epigenetic yomhlaba wonke phezu kokubonakaliswa kwezakhi zofuzo. Le ndlela isebenzisa i-transposase ye-Tn5 esebenzayo ukuze ihlukanise futhi ivule izifunda ze-chromatin ngasikhathi sinye ngokufaka ama-adaptha okulandelana. Ukwandiswa kwe-PCR okulandelayo kuphumela ekudalweni komtapo wolwazi wokulandelana, okuvumela ukuhlonza okuphelele kwezifunda ze-chromatin ezivulekile ngaphansi kwezimo ezithile zesikhala-sikhathi. I-ATAC-seq inikeza umbono ophelele wezindawo ze-chromatin ezifinyelelekayo, ngokungafani nezindlela ezigxila kuphela ezindaweni zokubopha izinto ezibhaliwe noma izifunda ezithile eziguqulwe yi-histone. Ngokulandelanisa lezi zifunda ze-chromatin ezivulekile, i-ATAC-seq yembula izifunda ezinamathuba amaningi okulandelana okusebenzayo okulawulayo kanye nezindawo zokubopha izinto ezibhaliwe ezingaba khona, okunikeza ukuqonda okubalulekile ekuguqulweni okuguquguqukayo kokubonakaliswa kwezakhi zofuzo kulo lonke i-genome.
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Ukwehliswa Kokumelwa Kwe-Bisulfite Sequencing (RRBS)
Ukwehliswa kokumelwa kwe-bisulfite sequencing (RRBS) kuncike ekucebiseni izifunda ezicebile eziqhingini ze-CpG ngokuhlukaniswa kwe-MspI kulandelwe ukukhethwa kosayizi wezingcezu ezingama-200-500/600 bps. Ngenxa yalokho, yizifunda eziseduze neziqhingi ze-CpG kuphela ezihlelwa ngokulandelana, kuyilapho lezo ezineziqhingi ze-CpG ezikude zingafakwanga ekuhlaziyweni. Le nqubo, ehlanganiswe nokwehliswa kwe-bisulfite sequencing, ivumela ukutholakala kwe-methylation ye-DNA enesinqumo esiphezulu, kanti indlela yokwehliswa kwe-sequencing, i-PE150, igxile ngqo emaphethelweni ezi-insert esikhundleni se-middle, okwandisa ukusebenza kahle kwe-methylation profiling.
Le ndlela ivele njengendlela engcono kakhulu futhi ephumelelayo esikhundleni se-Whole Genome Bisulfite Sequencing (WGBS) ocwaningweni lwe-DNA methylation. Ngenkathi i-WGBS inikeza ukuqonda okuphelele ngokuhlola lonke i-genome ngesisombululo esisodwa, izindleko zayo eziphakeme zingaba yisici esinqunyelwe, i-RRBS eyinciphisa ngokuhlelekile le nselele ngokuhlaziya ngokukhetha ingxenye emele i-genome. Le ndlela iyithuluzi elibalulekile elivumela ucwaningo lwe-DNA methylation olungabizi futhi luthuthukise ulwazi lwezindlela ze-epigenetic.
