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Single-nuclei RNA Sequencing
Kupanga makina ojambulira selo limodzi ndi njira zomangira laibulale yanthawi zonse, kuphatikizidwa ndi kutsatizana kopitilira muyeso, kwasintha maphunziro a jini pamaselo. Kupambana kumeneku kumapangitsa kusanthula mozama komanso mozama za kuchuluka kwa maselo ovuta, kuthana ndi malire omwe amakhudzana ndi kuchuluka kwa ma jini pama cell onse ndikusunga kusiyanasiyana kowona m'maguluwa. Ngakhale kutsatizana kwa selo imodzi ya RNA (scRNA-seq) kuli ndi ubwino wosatsutsika, kumakumana ndi zovuta m'magulu ena kumene kupanga kuyimitsidwa kwa selo limodzi kumakhala kovuta ndipo kumafuna zitsanzo zatsopano. Ku BMKGene, timathana ndi vutoli popereka nyukiliya imodzi ya RNA sequencing (snRNA-seq) pogwiritsa ntchito ukadaulo wapamwamba kwambiri wa 10X Genomics Chromium. Njirayi imakulitsa kuchuluka kwa zitsanzo zomwe zimatha kusanthula transcriptome pamlingo wa selo limodzi.
Kudzipatula kwa ma nuclei kumachitika kudzera mu chipangizo chamakono cha 10X Genomics Chromium, chokhala ndi makina asanu ndi atatu a microfluidics okhala ndi kuwoloka kawiri. Mkati mwa dongosololi, mikanda ya gel yomwe imaphatikizapo ma barcode, zoyambira, ma enzyme, ndi phata limodzi zimayikidwa mu madontho amafuta a nanoliter, kupanga Gel Bead-in-Emulsion (GEM). Kutsatira kupangidwa kwa GEM, cell lysis ndi barcode kumasulidwa kumachitika mkati mwa GEM iliyonse. Pambuyo pake, mamolekyu a mRNA amalembedwa m'ma cDNAs, kuphatikiza ma barcode 10X ndi Unique Molecular Identifiers (UMIs). Ma cDNA awa amasinthidwa kukhala laibulale yotsatizana, kuthandizira kuwunika kozama komanso kozama kwa mbiri ya jini pamlingo wa selo imodzi.
Pulatifomu: 10 × Genomics Chromium ndi Illumina NovaSeq Platform
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10x Genomics Visium Spatial Transcriptome
Spatial transcriptomics ndiukadaulo wotsogola womwe umalola ofufuza kuti afufuze momwe ma jini amafotokozera mkati mwa minofu ndikusunga malo awo. Pulatifomu imodzi yamphamvu mderali ndi 10x Genomics Visium yophatikizidwa ndi kutsatizana kwa Illumina. Mfundo ya 10X Visium yagona pa chip chapadera chokhala ndi malo ojambulidwa omwe zigawo za minofu zimayikidwa. Malo ogwidwawa ali ndi mabala a barcode, aliwonse ofanana ndi malo apadera mkati mwa minofu. Mamolekyu a RNA omwe adagwidwa kuchokera m'minyewayo amalembedwa ndi zozindikiritsa mamolekyulu (UMIs) panthawi yolemba mobwereza. Malo okhala ndi barcode awa ndi ma UMI amathandizira kupanga mapu olondola a malo komanso kuchuluka kwa mafotokozedwe a jini pakusintha kwa selo limodzi. Kuphatikiza kwa zitsanzo zokhala ndi barcode ndi UMIs zimatsimikizira kulondola ndi kutsimikizika kwa deta yopangidwa. Pogwiritsa ntchito ukadaulo uwu wa Spatial Transcriptomics, ofufuza amatha kumvetsetsa mozama momwe ma cell amapangidwira komanso momwe ma cell amagwirira ntchito mkati mwa minyewa, ndikupereka chidziwitso chamtengo wapatali pamakina omwe amachokera kuzinthu zachilengedwe m'magawo angapo, kuphatikiza oncology, neuroscience, biology yachitukuko, immunology. , ndi maphunziro a botanical.
Platform: 10X Genomics Visium ndi Illumina NovaSeq
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Kutalika Kwathunthu kwa mRNA Sequencing-Nanopore
Ngakhale kutsatizana kwa mRNA kochokera ku NGS ndi chida chosunthika chowerengera ma jini, kudalira kwake pamawerengedwe afupikitsa kumalepheretsa kugwira ntchito kwake pakuwunika zovuta za transcriptomic. Kumbali inayi, kutsatizana kwa nanopore kumagwiritsa ntchito ukadaulo wowerengera nthawi yayitali, zomwe zimathandizira kutsatizana kwa zolembedwa zazitali za mRNA. Njirayi imathandizira kufufuza mwatsatanetsatane njira zina zophatikizira, kuphatikiza ma jini, poly-adenylation, komanso kuchuluka kwa ma isoforms a mRNA.
Nanopore sequencing, njira yomwe imadalira ma signature amagetsi a nanopore single-molecule nthawi yeniyeni, imapereka zotsatira zenizeni zenizeni. Motsogozedwa ndi mapuloteni amagalimoto, DNA yamitundu iwiri imamangiriza ku mapuloteni a nanopore ophatikizidwa mu biofilm, kumasuka pamene ikudutsa munjira ya nanopore pansi pa kusiyana kwamagetsi. Zizindikiro zamagetsi zapadera zomwe zimapangidwa ndi maziko osiyanasiyana pa chingwe cha DNA zimazindikirika ndikugawidwa mu nthawi yeniyeni, zomwe zimathandizira kutsata kolondola komanso kosalekeza kwa nucleotide. Njira yatsopanoyi imagonjetsa malire owerengeka pang'ono ndipo imapereka nsanja yosinthika yowunikira ma genomic, kuphatikiza maphunziro ovuta a transcriptomic, ndi zotsatira zaposachedwa.
Platform: Nanopore PromethION 48
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Kutsata kutalika kwa mRNA -PacBio
Ngakhale kutsatizana kwa mRNA kochokera ku NGS ndi chida chogwiritsa ntchito mosiyanasiyana pakuwerengera ma jini, kudalira kwake pamawerengedwe afupikitsa kumalepheretsa kugwiritsidwa ntchito kwake pakuwunikira zovuta zolembera. Kumbali ina, PacBio sequencing (Iso-Seq) imagwiritsa ntchito ukadaulo wowerengera nthawi yayitali, zomwe zimathandizira kutsatizana kwa zolembedwa zazitali za mRNA. Njirayi imathandizira kufufuza mwatsatanetsatane njira zina zophatikizira, kuphatikiza ma gene, ndi poly-adenylation. Komabe, pali zisankho zina za gene expression quantification chifukwa cha kuchuluka kwa deta yofunikira. Ukadaulo wotsatizana wa PacBio umadalira kutsatizana kwa molekyulu imodzi, nthawi yeniyeni (SMRT), kupereka mwayi wapadera wojambula zolemba zazitali za mRNA. Njira yatsopanoyi imaphatikizapo kugwiritsa ntchito zero-mode waveguides (ZMWs) ndi zitsime zokhala ndi microfabricated zomwe zimathandiza kuti nthawi yeniyeni ya DNA polymerase iwonetsedwe panthawi yotsatizana. Mkati mwa ma ZMW awa, PacBio's DNA polymerase imapanga ulusi wowonjezera wa DNA, ndikupanga zowerengera zazitali zomwe zimatenga zolemba zonse za mRNA. Opaleshoni ya PacBio mu Circular Consensus sequencing (CCS) imakulitsa kulondola mwa kutsata mobwerezabwereza molekyulu yomweyo. Mawerengedwe a HiFi omwe amapangidwa ali ndi kulondola kofanana ndi NGS, zomwe zimathandizira pakuwunika kwatsatanetsatane komanso kodalirika kwa zovuta zolembera.
Platform: PacBio Sequel II; PacBio Revio
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Eukaryotic mRNA Sequencing-NGS
Tsatanetsatane wa mRNA, ukadaulo wosunthika, umathandizira kusanthula kwatsatanetsatane kwa zolembedwa zonse za mRNA m'maselo malinga ndi momwe zinthu ziliri. Ndi kugwiritsa ntchito kwake mosiyanasiyana, chida chotsogolachi chimavumbulutsa mbiri yamitundu yodabwitsa, kapangidwe ka majini, ndi njira zama cell zomwe zimagwirizanitsidwa ndi mitundu yosiyanasiyana yachilengedwe. Kutengera kwambiri pakufufuza kofunikira, kuwunika kwachipatala, komanso kakulidwe ka mankhwala, kutsatizana kwa mRNA kumapereka chidziwitso pazovuta zama cellular dynamics ndi genetic regulation, zomwe zimadzetsa chidwi cha kuthekera kwake m'magawo osiyanasiyana.
Platform: Illumina NovaSeq X; Chithunzi cha DNBSEQ-T7
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Non-Reference based mRNA Sequencing-NGS
Kutsatizana kwa mRNA kumapatsa mphamvu kusanthula kwatsatanetsatane kwa zolembedwa zonse za mRNA m'maselo malinga ndi momwe zinthu ziliri. Ukadaulo wotsogolawu umagwira ntchito ngati chida champhamvu, kuwulula mbiri yakale ya jini, kapangidwe ka majini, ndi njira zama cell zomwe zimagwirizanitsidwa ndi mitundu yosiyanasiyana yachilengedwe. Kutengera kwambiri pakufufuza kofunikira, zowunikira zamankhwala, komanso kakulidwe ka mankhwala, kutsatizana kwa mRNA kumapereka chidziwitso pazovuta zama cellular dynamics ndi genetic regulation.
Platform: Illumina NovaSeq X; Chithunzi cha DNBSEQ-T7
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Long Non-coding Sequencing-Illumina
Ma RNA autali osakhota zilembo (lncRNAs) ndiatali kuposa ma nucleotide 200 omwe ali ndi kuthekera kocheperako ndipo ndi zinthu zofunika kwambiri mkati mwa RNA yosalemba. Zopezeka mu nucleus ndi cytoplasm, ma RNA awa amatenga gawo lofunikira pakuwongolera epigenetic, transcriptal, ndi post-transcriptal, kutsimikizira kufunikira kwawo pakupanga ma cell ndi ma cell. Kutsata kwa LncRNA ndi chida champhamvu pakusiyanitsa ma cell, Ontogenesis, ndi matenda a anthu.
Platform: Illumina NovaSeq
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Small RNA Sequencing-Illumina
Mamolekyu ang'onoang'ono a RNA (sRNA), amaphatikiza ma microRNAs (miRNAs), ma RNA ang'onoang'ono osokoneza (siRNAs), ndi ma RNA ophatikizana ndi piwi (piRNAs). Mwa awa, ma miRNA, ozungulira 18-25 ma nucleotides aatali, ndiwodziwika kwambiri chifukwa cha maudindo awo ofunikira pamachitidwe osiyanasiyana am'manja. Pokhala ndi mawonekedwe okhudzana ndi minofu komanso siteji, ma miRNA amawonetsa kutetezedwa kwakukulu pamitundu yosiyanasiyana.
Platform: Illumina NovaSeq
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CircRNA Sequencing-Illumina
Zozungulira RNA sequencing (circRNA-seq) ndikuwonetsa ndi kusanthula ma RNA ozungulira, gulu la mamolekyu a RNA omwe amapanga malupu otsekedwa chifukwa cha zochitika zosagwirizana ndi canonical splicing, zomwe zimapangitsa kuti RNA iyi ikhale yokhazikika. Ngakhale ma circRNA ena awonetsedwa kuti amachita ngati masiponji a microRNA, kuthamangitsa ma microRNA ndikuwalepheretsa kuwongolera ma mRNAs awo, ma circRNA ena amatha kulumikizana ndi mapuloteni, kusintha mawonekedwe a jini, kapena kukhala ndi maudindo pama cell. Kusanthula kwa mawu a circRNA kumapereka chidziwitso pamaudindo owongolera mamolekyuwa komanso kufunika kwawo pama cell osiyanasiyana, kakulidwe, ndi matenda, zomwe zimathandizira kumvetsetsa mozama za kuvutikira kwa malamulo a RNA pamayendedwe a jini.
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Kutsata Kwathunthu kwa Transcriptome - Illumina
Kutsatizana kwa zolemba zonse kumapereka njira yokwanira yolembera mamolekyu osiyanasiyana a RNA, kuphatikiza ma coding (mRNA) ndi ma RNA osalemba (lncRNA, circRNA, ndi miRNA). Njirayi imagwira zolemba zonse zama cell enieni panthawi inayake, ndikupangitsa kuti timvetsetse bwino njira zama cell. Zomwe zimatchedwanso "total RNA sequencing," cholinga chake ndi kuwulula maukonde owongolera pamlingo wa transcriptome, kuthandizira kusanthula mozama monga kupikisana kwa RNA (ceRNA) ndi kusanthula kwa RNA. Izi ndizomwe zimayambira poyambira kuzindikiritsa magwiridwe antchito, makamaka pakuwulula maukonde owongolera omwe akuphatikizana ndi circRNA-miRNA-mRNA-based ceRNA.
