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I-Single- nuclei rna ukulandelana

I-Single- nuclei rna ukuhlelwa kabusha kwesithombe esifakiwe

Ukuthuthukiswa kokuthwebula kweseli elilodwa namasu okwakha umtapo wezincwadi, okuhambisana nokulandelana okuphezulu okuphezulu, kuguqule izifundo zezincazelo zofuzo ezingeni leseli. Lokhu kuphumelela kuvumela ukuhlaziya okujulile nokubanzi ngokwengeziwe kwama-cell cell. Ngenkathi ukuhlelwa kabusha kwe-RNA eyodwa (Scrna-SEQ) kunezinzuzo ezingenakuphikwa, kuhlangana nezinselelo zezicubu ezithile lapho ukusungulwa kokumiswa kweseli elilodwa kufakazela ubunzima futhi kudinga amasampula amasha. E-BMKGENE, sibhekana nalesi simangalo ngokunikeza ukulandelana kwe-single-nucleus rna (SNRNA-SEQ) kusetshenziswa ubuchwepheshe be-chromium yezwe. Le ndlela isondele i-spectrum yamasampula azenzeke ekuhlaziyweni okudluliselwe ku-cell cell.

Ukuhlukaniswa kwe-nuclei kwenziwa nge-10x genomics ye-10x genomics Chromium Chip, eneziteshi eziyisishiyagalombili ze-microfluidics nge-Double Crossnings. Ngaphakathi kwalolu hlelo, ubuhlalu be-gel buhlanza ama-barcode, ama-primers, ama-enzyme, kanye ne-nucleus eyodwa kungenelwe amaconsi kawoyela we-nanoliter, akha i-gel bead-in-emulsion (GEM). Ukulandela ukwakheka kwe-GEM, i-cell lysis kanye nokukhishwa kwebhakhodi kwenzeka ngaphakathi kwegugu ngalinye. Kamuva, ama-moleculernar molecule aphambukela emuva kwi-CDNas, okufaka ama-barcode ayi-10x kanye nezikhombi zamangqamuzana ezihlukile (i-UMIS). Lezi zi-CDN ziba ngaphansi kokwakhiwa kwezincwadi ezijwayelekile zokulandelana, okwenza ukuhlola okuqinile nokubukwa okuphelele kwamaphrofayili e-gene venvel ezingeni leseli elilodwa.

Ipulatifomu: 10 × genomics chromium kanye ne-Illina NovaseQ yesikhulumi

Xhumana nathi

Imininingwane ye-Service

Bioinformatics

Imiphumela yedemo

Izincwadi Ezifakiwe

Isikimu sobuchwepheshe

Ukuhlukaniswa kwe-nuclei kutholakala nge-10 × genomics Chromium ™, equkethe uhlelo lwesi-microfluidics eziyisishiyagalombili ngokuwela kabili. Kulesi simiso, ubuhlalu be-gel nama-barcode kanye nama-primer, ama-enzyme kanye ne-nzleus eyodwa kufakwa empompini kawoyela we-nanoliter, ekhiqiza i-gel bead-in-emulsion (GEM). Lapho nje i-GEM yakheka, i-cell lysis kanye nokukhishwa kwamabhakhodi kwenziwa ku-gem ngayinye. I-MRNA ibuyiselwe emuva kuma-molecule e-CDNA ngama-barcode ayi-10 × kanye ne-UMI, okuhlobene kakhulu nokwakhiwa kwetabhulali okujwayelekile.

Izici

● Ukulungiswa kokumiswa kwe-nuclei eyodwa kusuka ezicutshini eqandisiwe

● Ukwakheka kwe-gel bead-in-emulsion (GEM) elandelwa yi-CDNA synthesis

● Ubuhlalu ngalunye kwigugu lilayishwe ngama-primers akhiwa izigaba ezi-4:

i-poly (dt) umsila we-mrna priming kanye ne-cdna synthesis,

Isikhombi se-molecular (i-UMI) sokulungisa i-amplication bias

Ibhakhodi ye-10x

Ukulandelana okubophayo kokufunda okuyingxenye 1 ukulandelana kokulandelana

Izinzuzo

Ukulandelanisa okukodwa kwe-nucleus rna kuvimbela ukulinganiselwa kokulandelana kwe-RNA eyodwa, ukunika amandla:

● Ukusetshenziswa kwamasampula aqandisiwe futhi akugcini kumasampula amasha kuphela

● Ukucindezelwa okuphansi kwamaseli aqandisiwe uma kuqhathaniswa nokwelashwa kwe-enzymatic kwamaseli amasha, kuboniswe kwimininingwane edluliselwa ngohlobo lwezinhlobonhlobo zengcindezi

● Akunasidingo sokususwa kwangaphambili kwamangqamuzana abomvu egazi

● Ububanzi obungenamkhawulo weseli

● Amasampula amakhulu amasampula afanelekile ukuhlaziya, kufaka phakathi izinhlobo zezicubu eziyinkimbinkimbi futhi ezintekenteke ezithambekele kwiseli noma ekubhujisweni ngesikhathi sokuhlukaniswa kwezicubu

Amasampula angenakuhlaziywa ngokulandelana kwe-RNA ye-RNE-Cell

Iseli / izicubu	Isisusa
Izicubu eziqandisiwe zeqhwa	Ayikwazi ukuthola izinhlangano ezintsha noma ezinde ezigciniwe
Iseli yemisipha, iMegakaMayacyte, Amafutha ...	Ububanzi beseli bukhulu kakhulu ukufaka ithuluzi
Isibindi ...	Kube yintekenteke kakhulu ukuphula, ayikwazi ukuhlukanisa amaseli angashadile
Iseli le-Neuron, ubuchopho ...	Kubucayi kakhulu, kulula ukucindezela, kuzoshintsha imiphumela yokulandelana
Pancreas, i-thyroid ...	Ucebile ngama-enzyme endonous, okuthinta ukukhiqizwa kokumiswa kweseli elilodwa

Single-nucleus vs cell eyodwa

Single-nucleus	Iseli elilodwa
Ububanzi obungenamkhawulo weseli	Ububanzi be-Cell: 10-40 μm
Izinto ezibonakalayo zingaba yizicubu ezifriziwe	Okuqukethwe kumele kube yizicubu ezintsha
Ukucindezela okuphansi kwamaseli afriziwe	Ukwelashwa kwe-enzyme kungadala ukusabela kwengcindezi
Awekho amaseli abomvu adinga ukususwa	Amangqamuzana abomvu egazi adinga ukususwa
I-Nuclear iveza ama-bioiconform	Iseli lonke liveza i-bioiconform

Ukucacisa

Izidingo zesampula

Indlu ehlala izincwadi

Ukuhlehlisa Isu

Kunconywe idatha

Ikhwalithi yokulawula

Izicubu zezilwane ≥ 200 mg

Izitshalo izicubu ≥ 400 mg

I-10x genomics Sn CDNA Library

I-Illumina PE150

I-100K PE ifunda iseli ngalinye

(100-200 GB)

I-700-1200 nuclei / μL nobuqotho be-nuclei obuphawulwe ngaphansi kwesibonaka

Ngemininingwane engaphezulu ngesiqondisi sokulungiswa kwesampula kanye nokusebenza komsebenzi wesevisi, sicela ukhululeke ukukhuluma noBMKGENE Isazi

Ukugeleza komsebenzi wesevisi

Okwedlule: Isitshalo / isilwane sonke se-genome

Olandelayo: Ukuhlaziywa kwe-genome-reassociation association

Kubandakanya ukuhlaziywa okulandelayo:

● Ukulawulwa kwekhwalithi: Inani lamaseli, ukutholwa kweGene, Ukuhlonzwa Okunembile Kwamaseli, Ama-Molecule e-RNA NOKUFUNDA KAKHULU

● Ukuhlaziywa kwesampula yangaphakathi:

Ukuhlanganisa amaseli kanye nesichasiselo seqoqo

Ukuhlaziywa Kwezincazelo Ehlukile: Ukukhonjwa kwama-degs ngamaqoqo

Isichasiso esisebenzayo kanye nokucebisa kwama-degs weqembu

● Ukuhlaziywa kweqembu leqembu:

Inhlanganisela yedatha

Ukuhlaziywa Kwezimo Ezihlukile: Ukukhonjwa kwama-degs ngamaqembu

Isichasiso esisebenzayo kanye nokucebisa kwama-degs weqembu

● Ukuhlaziywa okuthuthukile:

Ukuhlaziywa Komjikelezo Weseli

Ukuhlaziywa kwe-Pseudotime

Ukuhlaziywa Kwe-Cell Communication (CellphonEDB)

UGene usetha ukuhlaziya ukucebisa (GSEA)

Ukuhlaziywa Kwangaphakathi Kwangaphakathi

Ukuhlangana kweseli:

Ukuhlaziywa Kwezimo Ezihlukile: Ama-Cluster Degs

Ukuhlaziywa kweqembu

Ukuhlaziywa Kwezimo Ezihlukile: Ama-Degs Eqembu

Ukuhlaziywa okuthuthukile:

Ukuhlaziywa kwe-Pseudotime:

Ukuhlaziywa komjikelezo weseli:

Hlola intuthuko eyenziwe nge-Bmkgene's Single-Nucleus RNA yokuhlehlisa izinsizakalo nge-10x chromium kulezi zincwadi ezifakiwe:

Wang, L. et al. (Ukuhlaziywa kwe-transcripcric okukodwa kuveza indawo yokuzivikela kwamaphaphu e-asthma-asthma aspoid-asthma aspoid asthma aspoid ',Ukuqhubeka kwe-National Academy of Science of I-United States of America, 118 (2), k. E2005590118. Doi: 10.1073 / PNAS.2005590118

Zheng, H. et al. (2022) 'Inethiwekhi yokulawula emhlabeni jikelele ye-Dyse vocurated voy expression kanye nokusayina okungajwayelekile kwe-metabolic kumaseli omzimba asezingeni eliphakeme le-micronves kanye ne-hashimoto's thyroiditis',Imingcele ku-immunology, 13, k. 879824. Doi: 10.3389 / Fimmu.2022.8799824 / Bibtex.

UTian, H. et al. (2023) 'I-Store-Cell cellOkwanyathela, 566, k. 739238. Doi: 10.1016 / J.Aquacilture.2023.739238.

Yu, Y. et al. (2023) I-PhotoDodynamic Therapy ithuthukisa umphumela wokuvuselela amasosha okuvuselela ama-inhibitors e-immune yokuvuselela kabusha i-anti-turmus ezigulini ezinomdlavuza wesisu ',Umdlavuza wesisu, 26 (5), PP. 798-813. Doi: 10.1007 / S10120-023-01409-X / metric.