Ulandelelwano lwe-RNA ye-nuclei enye
Inkqubo yoBugcisa
Ukuhlukaniswa kweenuclei kufezekiswa yi-10 × Genomics Chromium™, equlathe inkqubo ye-microfluidics yeendlela ezisibhozo ene-double crossings. Kule nkqubo, ii-gel beads ezinee-barcodes kunye ne-primer, ii-enzymes kunye ne-nucleus enye zifakwe kwi-nanoliter-size oil drop, nto leyo evelisa i-Gel Bead-in-Emulsion (GEM). Nje ukuba i-GEM yenziwe, i-cell lysis kunye nokukhululwa kwee-barcodes kwenziwa kwi-GEM nganye. I-mRNA iguqulelwa ngasemva kwii-molecules ze-cDNA ezinee-barcodes ze-10 × kunye ne-UMI, eziphantsi kolwakhiwo oluqhelekileyo lwelayibrari.
Iimbonakalo
● Ukulungiswa kwe-single-nuclei suspension evela kwizicubu eziqandisiweyo
● Ukwenziwa kwe-Gel Bead-in-Emulsion (GEM) kulandele ukwenziwa kwe-cDNA
● I-bead nganye kwi-GEM izaliswe zii-primer ezenziwe ngamacandelo ama-4:
umsila we-poly(dT) wokulungiselela i-mRNA priming kunye nokwenziwa kwe-cDNA,
I-Unique Molecular Identifier (UMI) yokulungisa i-amplification bias
Ibhakhowudi ye-10x
Ulandelelwano lokubopha lweprimer yokulandelelana yokufunda okuyingxenye 1
Iingenelo
Ulandelelwano lwe-RNA olune-nucleus enye luthintela imida yolandelelwano lwe-RNA oluneseli enye, nto leyo eyenza:
● Ukusetyenziswa kweesampuli ezikhenkcezisiweyo kwaye akupheleli nje kuphela kwiisampuli ezintsha
● Uxinzelelo oluphantsi lweeseli ezikhenkcezisiweyo xa kuthelekiswa nonyango lwe-enzyme lweeseli ezintsha, olubonakala kwidatha ye-transcriptome ngendlela yezakhi zofuzo ezibangelwa luxinzelelo oluncinci.
● Akukho mfuneko yokususa iiseli ezibomvu zegazi kwangaphambili
● Ububanzi beeseli obungenamda
● Uluhlu olukhulu lweesampuli ezifanelekileyo ukuhlalutywa, kuquka iintlobo zezicubu ezintsonkothileyo nezibuthathaka ezinokuba nefuthe lokuqhekeka okanye ukutshatyalaliswa kweeseli ngexesha lokuqhekeka kwezicubu
Iisampulu ezingenakuhlaziywa ngokulandelelanisa i-RNA yeseli enye kwaye zifanelekile ukulandelelanisa i-RNA ye-nuclei enye:
| Iseli/Ithishu | Isizathu |
| Ithishu eqandisiweyo entsha | Ayikwazi ukufumana imibutho emitsha okanye esele igcinwe ixesha elide |
| Iiseli zemisipha, iMegakaryocyte, Amafutha… | Ububanzi beseli bukhulu kakhulu ukuba bungangeni kwisixhobo |
| Isibindi… | Ibuthathaka kakhulu ukuba ingaqhekeka, ayikwazi ukwahlula iiseli ezizimeleyo |
| Iseli ye-Neuron, ubuchopho… | Ukuba novakalelo olungakumbi, kulula ukuxinzelela, kuya kutshintsha iziphumo zokulandelelana |
| I-Pancreas, i-thyroid… | Ityebile kwii-enzymes zendalo, ezichaphazela ukuveliswa kokumiswa kweseli enye |
I-nucleus enye vs i-Single-cell
| I-nucleus enye | Iseli enye |
| Ububanzi beeseli obungenamda | Ububanzi beseli: 10-40 μm |
| Le nto inokuba yilaphu eqandisiweyo | Izinto ezisetyenzisiweyo mazibe yilaphu entsha |
| Uxinzelelo oluphantsi lweeseli ezikhenkcezisiweyo | Unyango lwe-enzyme lunokubangela impendulo yoxinzelelo lweseli |
| Akukho ziseli zibomvu zegazi ekufuneka zisuswe | Iiseli ezibomvu zegazi kufuneka zisuswe |
| Inyukliya iveza ulwazi lwebhayoloji | Iseli yonke iveza ulwazi lwebhayoloji |
Iinkcukacha
| Iimfuno zeSampuli | Ithala leeNcwadi | Icebo lokulandelelanisa izinto | Idatha iyacetyiswa | Ulawulo lwemeko |
| Izicubu zezilwanyana ≥ 200 mg Izicubu zesityalo ≥ 400 mg | Ithala leencwadi le-10x Genomics sn cDNA | I-Illumina PE150 | Ukufundwa kwe-PE eyi-100K ngeseli nganye (100-200 Gb) | I-700-1200 nuclei/μl kunye nokuthembeka kwe-nuclei okubonwa phantsi kwe-microscope |
Ukuze ufumane iinkcukacha ezithe vetshe malunga nesikhokelo sokulungiselela isampuli kunye nendlela yokusebenza kwenkonzo, nceda uzive ukhululekile ukuthetha nomntuIngcali ye-BMKGENE
Ukuhamba komsebenzi wenkonzo
Kubandakanya uhlalutyo olulandelayo:
● Ulawulo loMgangatho: inani leeseli, ukufunyanwa kwezakhi zofuzo, ukuchongwa ngokuchanekileyo kweeseli, iimolekyuli ze-RNA kunye nokulinganiswa kokubonakaliswa
● Uhlalutyo lweSampulu yangaphakathi:
Ukuhlanganiswa kweeseli kunye nengcaciso yeqela
Uhlalutyo loKwahluka koKwahluka: ukuchongwa kwee-DEGs kumaqela
Isichazi-magama esisebenzayo kunye nokutyebisa ii-DEG zeqela
● Uhlalutyo phakathi kwamaqela:
Ukudibanisa idatha
Uhlalutyo loKwahluka koKwahluka: ukuchongwa kwee-DEG kumaqela
Isichazi-magama esisebenzayo kunye nokutyebisa ii-DEG zeqela
● Uhlalutyo oluPhambili:
Uhlalutyo lomjikelo weseli
Uhlalutyo lwexesha lobuxoki
Uhlalutyo lonxibelelwano lweselfowuni (iCellPhoneDB)
Uhlalutyo lokuFundiswa kweSeti yeZizalo (i-GSEA)
Uhlalutyo lwesampulu yangaphakathi
Ukuhlanganiswa kweeseli:
Uhlalutyo loKwahluka koKwahluka: ii-DEG zeqela
Uhlalutyo phakathi kwamaqela
Uhlalutyo loKwahluka koKwahluka: Ii-DEG zeQela
Uhlalutyo oluPhambili:
Uhlalutyo lwexesha lobuxoki:
Uhlalutyo lomjikelo weseli:
Hlola uphuhliso oluququzelelwe ziinkonzo ze-BMKGene's single-nucleus RNA sequencing yi-10X Chromium kwezi ncwadi zibalaseleyo:
UWang, L. et al. (2021) 'Uhlalutyo lwe-transcriptomic yeseli enye lubonisa imeko-bume yomzimba yokuzikhusela kwimiphunga kwi-asthma exacerbation exhathisayo i-steroid',Iingxelo zeNational Academy of Sciences yaseUnited States of America, 118(2), iphe. e2005590118. doi: 10.1073/pnas.2005590118
UZheng, H. et al. (2022) 'Inethiwekhi Yolawulo Lwehlabathi Lonke Yokubonakaliswa Kwezakhi Zofuzo Ezingasebenzi Kakuhle kunye Nokubonakaliswa KweeMetabolic Ezingaqhelekanga Kwiiseli Zomzimba Kwindawo Engqongileyo Yesifo SeeGraves kunye Nesifo Se-Thyroiditis SikaHashimoto',Imida kwi-Immunology, 13, iphe. 879824. doi: 10.3389/FIMMU.2022.879824/BIBTEX.
UTian, H. et al. (2023) 'I-transcriptome yeseli enye ityhila ukungafani kunye neempendulo zomzimba zee-leukocytes emva kokugonywa nge-Edwardsiella tarda engasebenziyo kwi-flounder (Paralichthys olivaceus)',Ukulima iintlanzi zasemanzini, 566, iphe. 739238. doi: 10.1016/J.AQUACULTURE.2023.739238.
Yu, Y. et al. (2023) 'Unyango lwe-Photodynamic luphucula iziphumo zezithinteli zokujonga amajoni omzimba ngokuguqula kwakhona amasosha omzimba alwa nomhlaza kwizigulana ezinomhlaza wesisu',Umhlaza Wesisu, 26(5), iphe. 798–813. doi: 10.1007/S10120-023-01409-X/METRICS.
