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BMKMANU S3000_Spatial Transcriptome
Spatial transcriptomics imatsogola pazatsopano zasayansi, kupatsa mphamvu ofufuza kuti afufuze movutikira momwe ma jini amafotokozera mkati mwa minofu ndikusunga malo awo. Pakati pa nsanja zosiyanasiyana, BMKGene yapanga BMKManu S3000 Spatial Transcriptome Chip, yomwe imadzitamandira ndi 3.5µm, yofikira pamagulu ang'onoang'ono, ndikupangitsa makonda amitundu yambiri. Chip cha S3000, chokhala ndi mawanga pafupifupi 4 miliyoni, chimagwiritsa ntchito tinthu tating'onoting'ono tomwe timakhala ndi mikanda yodzaza ndi zojambulidwa za barcode. Laibulale ya cDNA, yopangidwa ndi ma barcode a malo, imakonzedwa kuchokera ku chipangizo cha S3000 ndikutsatiridwa papulatifomu ya Illumina NovaSeq. Kuphatikiza kwa zitsanzo zokhala ndi barcode ndi UMIs zimatsimikizira kulondola ndi kutsimikizika kwa deta yopangidwa. Chip cha BMKManu S3000 ndi chosinthika kwambiri, chopereka zosintha zamitundu ingapo zomwe zimatha kusinthidwa bwino kumitundu yosiyanasiyana komanso tsatanetsatane watsatanetsatane. Kusinthasintha kumeneku kumayika chip ngati chisankho chabwino kwambiri pamaphunziro osiyanasiyana amtundu wapamalo, kuwonetsetsa kusanja kolondola kwa malo popanda phokoso lochepa. Kugwiritsa ntchito ukadaulo wogawa ma cell ndi BMKManu S3000 kumathandizira kugawa zolembera kumalire a maselo, zomwe zimapangitsa kusanthula komwe kuli ndi tanthauzo lachilengedwe. Kuphatikiza apo, kusintha kwabwino kwa S3000 kumabweretsa kuchuluka kwa majini ndi ma UMI omwe amapezeka pa selo iliyonse, zomwe zimapangitsa kusanthula kolondola kwambiri kwa mawonekedwe a malo osindikizira ndikuphatikizana kwa ma cell.
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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
