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Hi-C yochokera ku Chromatin Interaction
Hi-C ndi njira yomwe idapangidwa kuti ijambule kasinthidwe ka genomic pophatikiza kufufuza kuyanjana kochokera kufupi ndi kutsata kwapamwamba. Njirayi imachokera ku chromatin crosslinking ndi formaldehyde, kutsatiridwa ndi kugaya ndi kubwezeretsanso m'njira yoti tizidutswa tating'ono tating'ono tating'ono timene timagwirizanitsa timapanga katundu wa ligation. Posanja zinthu za ligation izi, ndizotheka kuphunzira gulu la 3D la genome. Hi-C imathandiza kuphunzira kugawidwa kwa magawo a genome omwe ali odzaza pang'ono (A compartments, euchromatin) ndipo amatha kukhala olembedwa bwino, ndi zigawo zomwe zimakhala zolimba kwambiri (B compartments, Heterochromatin). Hi-C itha kugwiritsidwanso ntchito kuzindikiritsa Topologically Associated Domains (TADs), zigawo za genome zomwe zimapindika ndipo zimatha kukhala ndi mawonekedwe ofanana, komanso kuzindikira malupu a chromatin, zigawo za DNA zomwe zimalumikizidwa pamodzi ndi mapuloteni komanso omwe ali. nthawi zambiri amalemeretsedwa muzinthu zowongolera. Ntchito yotsatizana ya BMKGene ya Hi-C imapatsa mphamvu ofufuza kuti afufuze kukula kwa ma genomics, ndikutsegula njira zatsopano zomvetsetsa malamulo a genome ndi zotsatira zake paumoyo ndi matenda.
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Chromatin Immunoprecipitation Sequencing (ChIP-seq)
Chromatin Immunoprecipitation (CHIP) ndi njira yomwe imathandizira ma antibodies kuti alemeretse mapuloteni omwe amamanga DNA ndi zolinga zawo zofananira. Kuphatikizika kwake ndi NGS kumathandizira kuti ma genome afotokozere za zolinga za DNA zomwe zimakhudzana ndi kusintha kwa histone, zinthu zolembera, ndi mapuloteni ena omwe amamanga DNA. Njira yosinthirayi imathandizira kufananitsa malo omangira pamitundu yosiyanasiyana yama cell, minofu, kapena mikhalidwe. Ntchito za ChIP-Seq zimachokera ku kuphunzira malamulo olembedwa komanso njira zachitukuko mpaka kuwunikira njira zamatenda, zomwe zimapangitsa kukhala chida chofunikira kwambiri pakumvetsetsa momwe ma genomic amayendera komanso kupititsa patsogolo chidziwitso chamankhwala.
Platform: Illumina NovaSeq
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Whole genome bisulfite sequencing (WGBS)
Whole Genome Bisulfite Sequencing (WGBS) imayima ngati njira yodziwika bwino ya golide yowunikira mozama DNA methylation, makamaka malo achisanu mu cytosine (5-mC), wowongolera kwambiri wamafotokozedwe amtundu ndi zochitika zama cell. Mfundo yomwe ili pansi pa WGBS imaphatikizapo chithandizo cha bisulfite, kupangitsa kutembenuka kwa ma cytosine osatulutsidwa kukhala uracil (C mpaka U), ndikusiya ma methylated cytosine osasinthika. Njirayi imapereka njira imodzi yokha, yomwe imalola ofufuza kuti afufuze mozama za methylome ndikupeza njira zachilendo za methylation zomwe zimagwirizanitsidwa ndi mikhalidwe yosiyanasiyana, makamaka khansa. Pogwiritsa ntchito WGBS, asayansi atha kupeza zidziwitso zosayerekezeka za mawonekedwe a genome-wide methylation, ndikupereka kumvetsetsa kwapang'onopang'ono kwa njira za epigenetic zomwe zimayambitsa njira zosiyanasiyana zamoyo ndi matenda.
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Kuyesa kwa Chromatin Yopezeka ndi Transposase yokhala ndi Sequencing High Throughput (ATAC-seq)
ATAC-seq ndi njira yotsatirira kwambiri yomwe imagwiritsidwa ntchito posanthula kupezeka kwa chromatin mu genome. Kugwiritsiridwa ntchito kumapereka kumvetsetsa kwakuya kwa machitidwe ovuta a epigenetic control pa gene expression. Njirayi imagwiritsa ntchito hyperactive Tn5 transposase kuti igawane ndikuyika magawo otseguka a chromatin poyika ma adapter otsatizana. Kukulitsa kwa PCR kotsatira kumapangitsa kuti pakhale laibulale yotsatizana, yomwe imalola kuzindikirika kwathunthu kwa zigawo zotseguka za chromatin pansi pamikhalidwe yanthawi yanthawi. ATAC-seq imapereka mawonekedwe athunthu amitundu yofikira ya chromatin, mosiyana ndi njira zomwe zimangoyang'ana pamasamba omangirira zinthu kapena zigawo zina zosinthidwa ndi histone. Posanjikiza zigawo zotseguka za chromatin, ATAC-seq imawulula madera omwe atha kukhala ndi katsatidwe kokhazikika komanso masamba omwe angamangirize, zomwe zimapereka chidziwitso chofunikira pakusinthika kwamtundu wa jini kudera lonselo.
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Kuchepetsa Kuyimilira kwa Bisulfite Sequencing (RRBS)
Reduced Representation Bisulfite Sequencing (RRBS) yatuluka ngati njira yotsika mtengo komanso yothandiza ku Whole Genome Bisulfite Sequencing (WGBS) mu kafukufuku wa DNA methylation. Ngakhale WGBS imapereka chidziwitso chokwanira powunika ma genome onse pamalingaliro amodzi, mtengo wake wokwera ukhoza kukhala wolepheretsa. RRBS imachepetsa vutoli mwa kusanthula mosankha gawo loyimira ma genome. Njirayi imadalira kulemeretsa kwa zigawo zolemera za zilumba za CpG ndi MspI cleavage yotsatiridwa ndi kusankha kukula kwa zidutswa za 200-500/600 bps. Chifukwa chake, madera okhawo omwe ali pafupi ndi zilumba za CpG amatsatiridwa, pomwe omwe ali ndi zilumba zakutali za CpG amachotsedwa pakuwunika. Njirayi, yophatikizidwa ndi kutsata kwa bisulfite, imalola kuzindikira kwapamwamba kwa DNA methylation, ndi njira yotsatizana, PE150, imayang'ana makamaka kumapeto kwa zoyikapo m'malo mwapakati, kukulitsa luso la methylation profiling. RRBS ndi chida chamtengo wapatali chomwe chimathandizira kufufuza kwa DNA methylation kopanda mtengo komanso kupititsa patsogolo chidziwitso cha njira za epigenetic.
