Ukulandelana kwe-RNA ye-nuclei eyodwa
Uhlelo Lobuchwepheshe
Ukuhlukaniswa kwama-nuclei kufezwa yi-10 × Genomics Chromium™, equkethe uhlelo lwe-microfluidics oluneziteshi eziyisishiyagalombili oluneziphambano ezimbili. Kulesi simiso, ubuhlalu bejeli obunama-barcode kanye ne-primer, ama-enzyme kanye ne-nucleus eyodwa buhlanganiswe ku-nanoliter-size oil drop, okukhiqiza i-Gel Bead-in-Emulsion (GEM). Uma i-GEM seyakhiwe, i-cell lysis kanye nokukhululwa kwama-barcode kwenziwa ku-GEM ngayinye. I-mRNA idluliselwa emuva ibe ama-molecule e-cDNA anama-barcode angu-10 × kanye ne-UMI, aphinde ancike ekwakhiweni komtapo wolwazi ojwayelekile.
Izici
● Ukulungiswa kokumiswa kwe-single-nuclei kusuka ezicutshini eziqandisiwe
● Ukwakheka kwe-Gel Bead-in-Emulsion (GEM) kulandelwe ukuhlanganiswa kwe-cDNA
● Ubuhlalu ngabunye ku-GEM bugcwele ama-primer akhiwe ngezigaba ezine:
umsila we-poly(dT) wokulungiselela i-mRNA kanye nokuhlanganiswa kwe-cDNA,
I-Unique Molecular Identifier (UMI) yokulungisa ukubandlulula kokukhulisa
Ibhakhodi engu-10x
Uchungechunge lokubopha lwe-primer yokulandelelana yokufunda okungaphelele engu-1
Izinzuzo
Ukulandelana kwe-RNA ye-single-nucleus kugwema ukulinganiselwa kokulandelana kwe-RNA yeseli elilodwa, okuvumela:
● Ukusetshenziswa kwamasampula aqandisiwe futhi akugcini nje kuphela kumasampula amasha
● Ukucindezeleka okuphansi kwamaseli aqandisiwe uma kuqhathaniswa nokwelashwa ngama-enzyme kwamaseli amasha, okubonakala kudatha ye-transcriptome ngesimo sezakhi zofuzo ezingabangelwa ukucindezeleka okukhulu.
● Akunasidingo sokususwa kwamangqamuzana abomvu egazi kusengaphambili
● Ububanzi beseli obungenamkhawulo
● Uhlu olukhulu lwamasampula afanelekela ukuhlaziywa, kufaka phakathi izinhlobo zezicubu eziyinkimbinkimbi nezibuthakathaka ezivame ukuhlangana noma ukubhujiswa kwamaseli ngesikhathi sokuhlukana kwezicubu
Amasampula angenakuhlaziywa ngokulandelelanisa i-RNA yeseli elilodwa futhi afanelekela ukulandelanisa i-RNA ye-nuclei eyodwa:
| Iseli/Izicubu | Isizathu |
| Izicubu eziqandisiwe ezingakavuthwa | Ayikwazi ukuthola izinhlangano ezintsha noma ezigcinwe isikhathi eside |
| Iseli lemisipha, i-Megakaryocyte, Amafutha… | Ububanzi beseli bukhulu kakhulu ukungena ethuluzini |
| Isibindi… | Ibuthakathaka kakhulu ukuba ingaphuka, ayikwazi ukuhlukanisa amaseli angawodwa |
| Iseli le-Neuron, Ubuchopho… | Ukuzwela kakhudlwana, okulula ukukucindezela, kuzoshintsha imiphumela yokulandelana |
| I-Pancreas, i-thyroid… | Icebile ngama-enzyme emvelo, ethinta ukukhiqizwa kokumiswa kweseli elilodwa |
I-nucleus eyodwa vs iseli elilodwa
| I-nucleus eyodwa | Iseli elilodwa |
| Ububanzi beseli obungenamkhawulo | Ububanzi beseli: 10-40 μm |
| Izinto zingaba yizicubu eziqandisiwe | Izinto ezisetshenziswayo kumele zibe yizicubu ezintsha |
| Ukucindezeleka okuphansi kwamaseli aqandisiwe | Ukwelashwa ngama-enzyme kungabangela ukusabela kokucindezeleka kwamaseli |
| Awekho amangqamuzana abomvu egazi okudingeka asuswe | Amaseli abomvu egazi adinga ukususwa |
| I-Nuclear iveza ulwazi lwe-bio | Iseli lonke liveza ulwazi lwe-bio |
Imininingwane
| Izidingo Zesampula | Umtapo Wolwazi | Isu lokulandelana kwezigaba | Kunconywa idatha | Ikhwalithi yokulawula |
| Izicubu zezilwane ≥ 200 mg Izicubu zesitshalo ≥ 400 mg | Umtapo wolwazi we-Genomics sn cDNA ongu-10x | I-Illumina PE150 | Ukufundwa kwe-PE okungu-100K ngeseli ngayinye (100-200 Gb) | 700-1200 nuclei/μl kanye nobuqotho benuclei obubonwa ngaphansi kwe-microscope |
Ukuze uthole imininingwane eyengeziwe mayelana nesiqondiso sokulungiselela isampula kanye nomsebenzi wesevisi, sicela ukhululeke ukukhuluma nochwepheshe.Uchwepheshe we-BMKGENE
Ukugeleza Komsebenzi Wesevisi
Kufaka phakathi ukuhlaziywa okulandelayo:
● Ukulawulwa Kwekhwalithi: inani lamaseli, ukutholwa kwezakhi zofuzo, ukuhlonza okunembile kwamaseli, ama-molecule e-RNA kanye nokulinganisa ukubonakaliswa
● Ukuhlaziywa Kwesampula Yangaphakathi:
Ukuhlanganiswa kwamaseli kanye nesichasiselo seqoqo
Ukuhlaziywa Kokuhlukaniswa Kokuhlukaniswa: ukuhlonza ama-DEG kumaqoqo
Isichasiselo esisebenzayo kanye nokucebisa ama-DEG eqembu
● Ukuhlaziywa kwamaqembu:
Inhlanganisela yedatha
Ukuhlaziywa Kokuhlukaniswa Kokuzibonakalisa: ukuhlonza ama-DEG ngamaqembu
Isichasiselo esisebenzayo kanye nokucebisa ama-DEG eqembu
● Ukuhlaziywa Okuthuthukisiwe:
Ukuhlaziywa komjikelezo weseli
Ukuhlaziywa kwesikhathi sokungafaneleki
Ukuhlaziywa kokuxhumana kweselula (i-CellPhoneDB)
Ukuhlaziywa Kokuthuthukiswa Kwesethi Yezakhi Zofuzo (i-GSEA)
Ukuhlaziywa kwesampula yangaphakathi
Ukuhlanganiswa kwamaseli:
Ukuhlaziywa Kokubonakaliswa Kokuhluka: ama-DEG eqembu
Ukuhlaziywa kwamaqembu
Ukuhlaziywa Kokubonakaliswa Kokuhluka: Ama-DEG Eqembu
Ukuhlaziywa Okuthuthukisiwe:
Ukuhlaziywa kwesikhathi sokungafaneleki:
Ukuhlaziywa komjikelezo weseli:
Hlola intuthuko eyenziwe yizinsizakalo zokulandelana kwe-RNA ze-single-nucleus RNA ze-BMKGene yi-10X Chromium kulezi zincwadi ezivelele:
UWang, L. et al. (2021) 'Ukuhlaziywa kwe-transcriptomic yeseli elilodwa kwembula isimo somzimba sokuzivikela samaphaphu ekukhulisweni kwe-asthma okumelana nama-steroid',Imibiko ye-National Academy of Sciences yase-United States of America, 118(2), ikhasi e2005590118. doi: 10.1073/pnas.2005590118
UZheng, H. et al. (2022) 'Inethiwekhi Yomhlaba Wonke Yokulawula Ukuvezwa Kwezakhi Zofuzo Okungahlelekile kanye Nokubonakaliswa Okungavamile Kwe-Metabolic Kumaseli Okuzivikela Emzimbeni Emvelweni Yezifo Zamathuna kanye ne-Hashimoto's Thyroiditis',Imingcele ku-Immunology, 13, ikhasi. 879824. doi: 10.3389/FIMMU.2022.879824/BIBTEX.
UTian, H. et al. (2023) 'I-transcriptome yeseli elilodwa iveza ukungafani kanye nezimpendulo zomzimba zama-leukocyte ngemva kokugonywa nge-Edwardsiella tarda engasebenzi ku-flounder (Paralichthys olivaceus)',Ukufuya izinhlanzi, 566, ikhasi 739238. doi: 10.1016/J.AQUACULTURE.2023.739238.
Yu, Y. et al. (2023) 'Ukwelashwa nge-photodynamic kuthuthukisa umphumela wezithiyo zokuhlola amasosha omzimba ngokuvuselela amasosha omzimba alwa nomdlavuza ezigulini ezinomdlavuza wesisu',Umdlavuza Wesisu, 26(5), amakhasi 798–813. doi: 10.1007/S10120-023-01409-X/METRICS.
