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Hal-nuklei RNA Taxane

Horumarinta qabsashada hal unug iyo farsamooyinka dhismaha maktabadda caadiga ah, oo ay weheliso isku xigxiga-sare, ayaa wax ka beddelay daraasadaha muujinta hidda-wadaha ee heerka unugga. Horumarkaani wuxuu ogolaanayaa in si qoto dheer oo qoto dheer loo falanqeeyo tirada unugyada adag, ka gudubka xaddidaadyada la xidhiidha celceliska muujinta hidda-wadaha ee dhammaan unugyada iyo ilaalinta kala duwanaanshaha dhabta ah ee dadkan. Iyadoo isku xigxiga hal unug ee RNA (scRNA-seq) uu leeyahay faa'iidooyin aan la dafiri karin, waxay la kulmeysaa caqabado unugyo gaar ah halkaas oo abuurista hal unug ka-joojin ay caddeyso mid adag oo u baahan muunado cusub. Markay tahay BMKGene, waxaanu wax ka qabanaynaa caqabadan annagoo bixinayna isku xigxiga hal-nucleus RNA (snRNA-seq) anagoo adeegsanayna tignoolajiyada casriga ah ee 10X Genomics Chromium. Habkani waxa uu balaadhinayaa shaybaarrada kala duwan ee lagu heli karo falanqaynta qoraalka ee heerka hal unug.

Go'doominta nuclei-yada waxaa lagu dhammeeyaa hal-abuurka 10X Genomics Chromium chip, oo leh nidaam siddeed kanaal ah oo microfluidics ah oo leh laba isgoys. Nidaamkan dhexdiisa, kuullaha jeelka ah ee ku jira barcodes, primers, enzymes, iyo hal nukleus ayaa ku jira dhibcaha saliidda ee nanoliter-ka, samaynta Gel Bead-in-Emulsion (GEM). Samaynta GEM ka dib, lysis unug iyo siidaynta barcode waxay ku dhacdaa GEM kasta. Ka dib, molecules mRNA waxay maraan qoraal kale oo cDNAs ah, oo lagu daro 10X barcodes iyo Aqoonsiyada Molecular Unique (UMIs). CDNA-yadan ayaa markaa la marsiiyaa dhismo isku xigxig oo caadi ah, taasoo fududaynaysa sahaminta adag oo dhamaystiran ee astaanta muujinta hidda-wadaha ee heerka hal unug.

Platform: 10× Genomics Chromium iyo Illumina NovaSeq Platform

Nala Soo Xidhiidh

Faahfaahinta Adeegga

Bioinformatics

Natiijooyinka Demo

Daabacaada sifada

Qorshaha Farsamada

Go'doominta nuclei-yada waxaa lagu gaaraa 10 × Genomics Chromium ™, kaas oo ka kooban siddeed kanaal oo ah nidaamka microfluidics oo leh laba isgoys. Nidaamkan, boodhadhka jel ee leh barcodes iyo primer, enzymes iyo hal nucleus ayaa ku jira dhibicda saliidda ee nanoliter-ka, taasoo dhalinaysa Gel Bead-in-Emulsion (GEM). Marka GEM la sameeyo, lysis unug iyo sii deynta barcodes ayaa lagu sameeyaa GEM kasta. mRNA waxa loo rogaa unugyo cDNA ah oo wata 10x barcodes iyo UMI, kuwaas oo ku sii xidhan dhismaha maktabadda isku xigxiga ee caadiga ah.

Astaamaha

● Diyaarinta ka joojinta hal-nuklei ee unugyada la qaboojiyey

Samaynta Gel Bead-in-Emulsion (GEM) oo ay ku xigto isku-darka cDNA

● Kuul kasta oo ku jira GEM-ka waxaa lagu raray asal-raacyo ka kooban 4 qaybood:

dabada poly(dT) ee mRNA priming iyo cDNA synthesis,

Aqoonsiga Molecular Unique (UMI) si loo saxo eexda

10x barcode

Isku-xigta isku-xiran ee akhrinta qayb ahaan 1 tix-raac ee aasaasiga ah

Faa'iidooyinka

Taxanaha RNA hal-nukleus ah waxa uu hareer maraa xaddidaadda isku xigxiga hal unug ee RNA, awoodna u siinaya:

● Isticmaalka muunado la qaboojiyey oo aan ku koobnayn oo keliya muunado cusub

● Walaaca hoose ee unugyada barafaysan marka la barbar dhigo daawaynta enzymatic ee unugyo cusub, oo ka muuqda xogta transcriptome ee qaabka hiddo-wadaha walaaca keena

● Looma baahna in ka hor laga saaro unugyada dhiigga cas

● Dhexroor unug aan xadidneyn

● Muunado badan oo u qalma falanqaynta, oo ay ku jiraan noocyada unugyada jilicsan iyo kuwa jilicsan kuwaas oo u nugul burburinta unugyada

Muunado aan lagu falanqeyn karin isku xigxiga hal unug oo RNA ah oo u qalma isku xigxiga hal unug ee RNA:

Unug / unug	Sababta
Ka saar unug barafaysan	Aan awoodin in la helo ururo cusub ama muddo dheer la badbaadiyey
Unugga murqaha, Megakaryocyte, Dufan…	Dhexroorka unuggu aad buu u weyn yahay si aan loo gelin qalabka
Beerka…	Aad bay u jilicsan tahay in la jebiyo, oo aan awoodin in la kala saaro unugyada keliya
Unugga neerfaha, Maskaxda…	Aad u xasaasisan, oo ay fududahay in la caddeeyo, ayaa bedeli doonta natiijooyinka isku xigxiga
Pankres, Thyroid…	Qani ku ah enzymes endogenous, oo saameeya soo saarista ka joojinta hal unug

Hal-nukleus vs Single-unug

Hal-xudun	Hal unug
Dhexroor unug aan xadidnayn	Dhexroorka unugga: 10-40 μm
Maaddadu waxay noqon kartaa unug barafaysan	Walaxdu waa inay noqotaa unug cusub
Walwalka hoose ee unugyada barafaysan	Daawaynta enzyme waxay keeni kartaa dareen-celinta cadaadiska unugga
Looma baahna in unugyada dhiigga cas laga saaro	Unugyada dhiigga cas waxay u baahan yihiin in la saaro
Nukliyeerku wuxuu muujiyaa macluumaadka noolaha	Unugga oo dhan waxa uu muujiyaa macluumaadka noolaha

Tilmaamaha

Shuruudaha Tusaalaha

Maktabadda

Istaraatiijiyada isku xigxiga

Xogta lagu taliyay

Xakamaynta tayada

Nudaha xoolaha ≥ 200 mg

Nudaha dhirta ≥ 400 mg

10x Genomics sn cDNA maktabadda

Illumina PE150

100k PE ayaa akhriya unug kasta

(100-200 Gb)

700-1200 nuclei/μl iyo hufnaanta xudunta oo lagu arkay mikroskoob

Faahfaahin dheeraad ah oo ku saabsan hagida diyaarinta muunada iyo socodka shaqada, fadlan xor u noqo inaad la hadasho aKhabiir BMKGENE

Socodka Shaqada Adeegga

Kii hore: Isku-xiridda Hiddo-sidaha Dhan ee Dhirta/Xayawaanka

Xiga: Falanqaynta Ururka Genom-ballaaran

Waxa ku jira falanqaynta soo socota:

● Xakamaynta tayada: tirada unugyada, ogaanshaha hidda-wadaha, aqoonsiga saxda ah ee unugyada, molecules RNA iyo cabirida hadalka

● Falanqaynta Muunada Gudaha:

Koox-kooxeed unug iyo sharraxaad kooxeed

Falanqaynta Muujinta Kala Duwan: aqoonsiga DEGs ee kutlada

Tilmaanta shaqaynaysa iyo tayeynta DEG-yada kutlada

● Falanqaynta kooxaha dhexdooda:

Isku darka xogta

Falanqaynta Muujinta Kala Duwan: aqoonsiga DEGs ee kooxo

Tilmaanta shaqaynaysa iyo kobcinta DEG-yada kooxda

● Falanqaynta Sare:

Falanqaynta wareegga unugyada

Falanqaynta wakhtiga beenta ah

Falanqaynta isgaarsiinta gacanta (CellPhoneDB)

Falanqaynta Kobcinta Gene Set (GSEA)

Falanqaynta muunada gudaha

Kooxda unugga:

Falanqaynta Muujinta Kala Duwan: DEGs-kutlada

Falanqaynta kooxaha dhexdooda

Falanqaynta Hadalka Kala Duwan: Kooxda DEGs

Falanqaynta Sare:

Falanqaynta wakhtiga beenta ah:

Falanqaynta wareegga unugyada:

Ku sahamin horumarka ay fududaysay BMKGene adeegyadeeda isku xigxiga hal-nucleus RNA ee 10X Chromium ee daabacadahan sifaysan:

Wang, L. iyo al. (2021) 'Falanqaynta transcriptomic-ka-hal-unuggu waxay daaha ka qaadaysaa muuqaalka difaaca sambabada ee neefta u adkeysata ee steroid',Talaabada Akademiyada Qaranka ee Sayniska ee Maraykanka, 118 (2), b. e2005590118. doi: 10.1073/pnas.2005590118

Zheng, H. iyo al. (2022) 'Shabakadda Nidaaminta Caalamiga ah ee Shabakadda Hiddaha ee Habacsanaansan iyo Calaamadaynta Dheef-shiid kiimikaadka Aan Caadiga ahayn ee Unugyada Difaaca ee Deegaanka Microe ee Cudurka Qabriga iyo Thyroiditis Hashimoto',Xuduudaha Immunology, 13, p. 879824. doi: 10.3389/FIMMU.2022.879824/BIBTEX.

Tian, H. iyo al. (2023) 'Transcriptome-ka-hal-unuggu waxay daaha ka qaadaysaa kala duwanaanshaha iyo jawaabaha difaaca ee leukocytes ka dib tallaalka Edwardsiella tarda aan firfircoonayn ee flounder (Paralichthys olivaceus)',Aquaculture, 566, b. 739238. doi: 10.1016/J.AQUACULTURE.2023.739238.

Yu, Y. iyo al. (2023) 'Daawaynta sawir-qaadista waxay wanaajisaa natiijada ka-hortagga isbaarada difaaca iyada oo dib-u-habayn ku samaynaysa difaaca ka-hortagga burooyinka ee bukaannada qaba kansarka caloosha',Kansarka caloosha, 26(5), bogga 798-813. doi: 10.1007/S10120-023-01409-X/METRICS.