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Kutsata kwa RNA ya nuclei imodzi
Kupanga njira zogwirira ntchito limodzi ndi laibulale yapadera, pamodzi ndi njira zamakono zogwirira ntchito, kwasintha maphunziro okhudza majini pamlingo wa selo. Kupambana kumeneku kumalola kusanthula mozama komanso mokwanira kwa kuchuluka kwa maselo ovuta, kuthana ndi zolepheretsa zomwe zimagwirizanitsidwa ndi kuchuluka kwa majini pa maselo onse ndikusunga kusiyana kwenikweni mkati mwa anthuwa. Ngakhale kuti kutsata kwa RNA ya selo limodzi (scRNA-seq) kuli ndi ubwino wosatsutsika, kumakumana ndi zovuta m'maselo ena pomwe kupanga kuyimitsidwa kwa selo limodzi kumakhala kovuta ndipo kumafuna zitsanzo zatsopano. Ku BMKGene, timathetsa vutoli popereka kutsata kwa RNA ya nucleus imodzi (snRNA-seq) pogwiritsa ntchito ukadaulo wapamwamba wa 10X Genomics Chromium. Njirayi imakulitsa mitundu yosiyanasiyana ya zitsanzo zomwe zingagwiritsidwe ntchito kusanthula kwa transcriptome pamlingo wa selo limodzi.
Kulekanitsidwa kwa ma nuclei kumachitika kudzera mu chipangizo chatsopano cha 10X Genomics Chromium chip, chokhala ndi njira ya microfluidics ya njira zisanu ndi zitatu yokhala ndi ma double crossings. Mkati mwa dongosololi, mikanda ya gel yokhala ndi ma barcode, ma primer, ma enzyme, ndi nucleus imodzi imayikidwa mu nanoliter-size oil drops, ndikupanga Gel Bead-in-Emulsion (GEM). Pambuyo pa kupangidwa kwa GEM, cell lysis ndi barcode release zimachitika mkati mwa GEM iliyonse. Pambuyo pake, ma mRNA molecules amadutsa reverse transcription kukhala ma cDNA, kuphatikiza ma barcode 10X ndi Unique Molecular Identifiers (UMIs). Ma cDNA awa amayikidwa mu standard sequencing library building, zomwe zimathandiza kufufuza bwino ma gene expression profiles pa single-cell level.
Pulatifomu: 10 × Genomics Chromium ndi Illumina NovaSeq Pulatifomu
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Kutsatana kwa mRNA kwa Utali Wonse
Ngakhale kuti kusanthula kwa mRNA kochokera ku NGS ndi chida chogwiritsidwa ntchito kwambiri powerengera kuchuluka kwa majini, kudalira kwake kuwerenga kwachidule kumalepheretsa kugwira ntchito kwake mu kusanthula kovuta kwa transcriptomic. Kumbali inayi, kusanthula kwa nanopore kumagwiritsa ntchito ukadaulo wowerenga nthawi yayitali, zomwe zimathandiza kusanthula kwa zolemba zonse za mRNA. Njirayi imathandizira kufufuza kwathunthu kwa njira zina zolumikizirana, kuphatikiza majini, poly-adenylation, ndi kuwerengera kwa isoforms za mRNA.
Kutsatana kwa nanopore, njira yomwe imadalira zizindikiro zamagetsi za nanopore imodzi-molekyulu yeniyeni, imapereka zotsatira munthawi yeniyeni. Motsogozedwa ndi mapuloteni oyendetsa magalimoto, DNA yokhala ndi zingwe ziwiri imalumikizana ndi mapuloteni a nanopore omwe ali mu biofilm, ndikumasuka pamene ikudutsa mu nanopore channel pansi pa kusiyana kwa magetsi. Zizindikiro zamagetsi zosiyana zomwe zimapangidwa ndi maziko osiyanasiyana pa chingwe cha DNA zimapezeka ndikugawidwa munthawi yeniyeni, zomwe zimapangitsa kuti nucleotide itsatizane molondola komanso mosalekeza. Njira yatsopanoyi imagonjetsa zoletsa zowerengera mwachidule ndipo imapereka nsanja yosinthika yowunikira majini ovuta, kuphatikiza maphunziro ovuta a transcriptomic, ndi zotsatira zachangu.
Nsanja: Nanopore PromethION 48
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Kutsata kwa mRNA kwautali wonse -PacBio
Ngakhale kusanthula kwa mRNA kochokera ku NGS ndi chida chogwiritsidwa ntchito kwambiri powerengera kuchuluka kwa majini, kudalira kwake kuwerenga kwachidule kumalepheretsa kugwiritsidwa ntchito kwake mu kusanthula kovuta kwa transcriptomic. Kumbali ina, kusanthula kwa PacBio (Iso-Seq) kumagwiritsa ntchito ukadaulo wowerenga nthawi yayitali, zomwe zimathandiza kusanthula kwa zolemba za mRNA nthawi yayitali. Njira iyi imathandizira kufufuza kwathunthu kwa kusakaniza kwina, kusakanikirana kwa majini, ndi poly-adenylation. Komabe, pali zosankha zina zowerengera kuchuluka kwa ma gene chifukwa cha kuchuluka kwa deta komwe kumafunika. Ukadaulo wowerengera wa PacBio umadalira kusanthula kwa ma molecule amodzi, nthawi yeniyeni (SMRT), zomwe zimapereka mwayi wapadera pakujambula zolemba za mRNA nthawi yayitali. Njira yatsopanoyi imaphatikizapo kugwiritsa ntchito ma waveguides a zero-mode (ZMWs) ndi zitsime zazing'ono zomwe zimathandiza kuwona nthawi yeniyeni ya ntchito ya DNA polymerase panthawi yowerengera. Mkati mwa ma ZMW awa, DNA polymerase ya PacBio imapanga chingwe chowonjezera cha DNA, ndikupanga kuwerenga kwakutali komwe kumaphatikizapo zolemba zonse za mRNA. Kugwira ntchito kwa PacBio mu Circular Consensus sequencing (CCS) mode kumawonjezera kulondola mwa kutsata molekyulu yomweyo mobwerezabwereza. Ma HiFi reads opangidwa ali ndi kulondola kofanana ndi NGS, zomwe zimathandizanso kusanthula kwathunthu komanso kodalirika kwa zinthu zovuta za transcriptomic.
Platform: PacBio Sequel II; PacBio Revio
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Kutsatana kwa mRNA ya Eukaryotic-NGS
Kutsata mRNA ndi njira yomwe imagwiritsa ntchito kutsata kwa m'badwo wotsatira kuti iwulule kupezeka ndi kuchuluka kwa mamolekyu a RNA mu chitsanzo cha zamoyo, kupereka chithunzithunzi cha momwe majini amaonekera mu chitsanzocho.
Ndi ntchito zake zosiyanasiyana, chida chamakono ichi chikuwonetsa mbiri zovuta za majini, kapangidwe ka majini, ndi njira zamamolekyu zogwirizana ndi njira zosiyanasiyana zamoyo.
Njira imeneyi imagwiritsidwa ntchito kwambiri mu kafukufuku, matenda azachipatala, komanso kupanga mankhwala, chifukwa imapereka chidziwitso cha zovuta za mphamvu zamaselo ndi malamulo a majini, zomwe zimapangitsa kuti anthu azifuna kudziwa zambiri zokhudza kuthekera kwake m'magawo osiyanasiyana.
Mapulatifomu omwe alipo: Illumina NovaSeq X; DNBSEQ-T7
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Kutsata kwa mRNA kochokera ku Non-Reference-NGS
Kutsata mRNA kumathandizira kusanthula kwathunthu kwa zolemba zonse za mRNA mkati mwa maselo pansi pa mikhalidwe inayake. Ukadaulo wamakono uwu umagwira ntchito ngati chida champhamvu, kuwulula mbiri zovuta za majini, kapangidwe ka majini, ndi njira zamamolekyu zogwirizana ndi njira zosiyanasiyana zamoyo. Kugwiritsidwa ntchito kwambiri mu kafukufuku woyambira, kuzindikira matenda, ndi chitukuko cha mankhwala, kutsata mRNA kumapereka chidziwitso cha zovuta za mphamvu zamaselo ndi malamulo a majini.
Pulatifomu: Illumina NovaSeq X; DNBSEQ-T7
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Kutsatana Kwautali Kosagwiritsa Ntchito Ma Code-Illumina
Ma RNA aatali osalemba ma code (lncRNAs) ndi aatali kuposa ma nucleotide 200 omwe ali ndi mphamvu yochepa yolemba ma code ndipo ndi zinthu zofunika kwambiri mkati mwa RNA yosalemba ma code. Amapezeka mu nyukiliyasi ndi cytoplasm, ma RNA awa amachita ntchito zofunika kwambiri mu epigenetic, transcriptional, ndi post-transcriptional regulation, zomwe zimatsimikizira kufunika kwawo pakupanga njira zama cell ndi ma molecular. Kutsata kwa LncRNA ndi chida champhamvu pakusiyanitsa maselo, Ontogenesis, ndi matenda a anthu.
Nsanja: Illumina NovaSeq X
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Kusanthula kwa RNA Yaing'ono-Illumina
RNA yaying'ono ndi gulu la mamolekyu a RNA omwe amaphatikizapo ma microRNA (miRNA), ma RNA ang'onoang'ono osokoneza (siRNA), ndi ma RNA olumikizana ndi piwi (piRNA). Pakati pa izi, ma miRNA, okhala ndi ma nucleotide pafupifupi 18-25, ndi odziwika bwino chifukwa cha ntchito zawo zofunika kwambiri pakulamulira machitidwe osiyanasiyana a maselo. Ndi mawonekedwe ake enieni a minofu ndi siteji, ma miRNA amasungidwa bwino kwambiri pamitundu yosiyanasiyana.
Pulatifomu: Illumina NovaSeq
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Kutsata kwa CircRNA-Illumina
Kusanthula kwa RNA yozungulira (circRNA-seq) kumalemba ndikuwunika ma RNA ozungulira, gulu la mamolekyu a RNA omwe amapanga ma loops otsekedwa chifukwa cha zochitika zosavomerezeka, zomwe zimapangitsa kuti RNA iyi ikhale yolimba kwambiri. Ngakhale ma circRNA ena awonetsedwa kuti amagwira ntchito ngati masiponji a microRNA, akumagawa ma microRNA ndikuletsa kuti asayang'anire ma mRNA awo omwe akufuna, ma circRNA ena amatha kuyanjana ndi mapuloteni, kusintha mawonekedwe a majini, kapena kukhala ndi maudindo mu njira zamaselo. Kusanthula kwa mawonekedwe a circRNA kumapereka chidziwitso pa ntchito zowongolera za mamolekyu awa ndi kufunika kwawo m'njira zosiyanasiyana zamaselo, magawo okukula, ndi matenda, zomwe zimathandiza kumvetsetsa bwino zovuta za malamulo a RNA pankhani yokhudza mawonekedwe a majini.
Pulatifomu: Illumina Novaseq X
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Kutsatana Kwathunthu kwa Transcriptome - Illumina
Kutsata njira ya transcriptome ya Wole kumapereka njira yokwanira yopezera ma molecule osiyanasiyana a RNA, kuphatikizapo ma coding (mRNA) ndi ma RNA osalemba ma code (lncRNA, circRNA, ndi miRNA). Njirayi imagwira transcriptome yonse ya maselo enaake panthawi inayake, zomwe zimathandiza kumvetsetsa bwino momwe maselo amagwirira ntchito.
Mapulatifomu omwe alipo: Illumina Novaseq X
