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I-nuclei ye-nuclei rna

Ukuphuhliswa kwesel-yeselfowuni kunye neendlela zokwakha zethala leencwadi, zidityaniswe nendlela ephezulu yokufaka, inesifundo se-telpored gestiction kwinqanaba leseli. Olu hlaziyo lo luvumela uhlalutyo olunzulu nolubanzi olubanzi lwezinto ezixineneyo zeseli, ukoyisa imiqobo enxulumene nokubhena kwi-gene ehambelana nazo zonke iiseli kunye nokugcina i-heterogeneity yokwenyani ngaphakathi kwaba bantu. Ngelixa i-screnimed ye-RNA ye-RNA (i-Scrna-Seq) ineenzuzo ezingathandekiyo, ihambelana nemiceli mngeni kwizicubu ezithile apho indalo yokumiswa kweeseli) isebenza nzima kwaye ifuna iisampulu ezitsha. EBmkgenene, sijongana nale ngxaki ngokubonelela nge-nucleus rna ye-nucleus ry Le ndlela ikhala i-spectrium yeesampulu ezinokuthi zingakwazi ukuthwala uhlalutsho kwindawo enye.

Ukuhlukaniswa kwe-nuclei kuyafezekiswa nge-10x ye-10x ye-10x ye-chip, ebonisa inkqubo yesibhozo ye-microfluidics yesitayile esinemimandla enqumlayo. Ngaphakathi kwale nkqubo, iibhlukhwe zejeli ezibandakanya i-barcode, i-Enzymes, kunye ne-enzymes, kunye ne-nucleus enye zine-lealine ye-e-Oliter-ratiber ye-e-Oliter, ye-Gel Bead-in-emulsion (GEM). Ukulandela ukwenziwa kweGEM, iseli yeylisi kunye nokukhutshwa kwebhakhowudi yenzeka kwi-gem nganye. Emva koko, i-MRNA Molecules iphinde ithunyelwe kwi-CDNAs, ibandakanya i-10x barcodes kunye ne-I-UMECURE yemolekyuli eyahlukileyo (i-IISs). Ezi cdnas ziphantsi ke zolwakhiwo olusemgangathweni lwethala leencwadi, ziququzelela ukuphononongwa okuqinileyo kunye nokuhlolisisa okuphezulu kweeprofayili ze-full kwinqanaba leseli elinye.

Iqonga: 10 × i-chromium ye-chromium kunye ne-Illina Novaseq

Qhagamshelana nathi

Iinkcukacha zenkonzo

I-Bioinformatics

Iziphumo

Ushicilelo olubonisiweyo

Iskimu sobugcisa

Ukuhlukaniswa kwe-nuclei kufezekisiwe ngu-10 × i-chromium ™, kubandakanya inkqubo ye-microfluidics yesibhozo yemicrofluidics ehamba ngeenyawo. Kule nkqubo, iibhlukhwe zejeli ngebhakhowudi kunye ne-primer, i-enzymes kunye ne-nucleus ye-enelite-hlue-hlue-i-e-I-Enoliter-Spred-In-Ead-in-emulsion (GEM). Nje ukuba i-GEM yenziwa, iseli yeylisi kunye nokukhululwa kwebhakhowudi kwenziwa kwi-gem nganye. I-MRNA ingabhalwanga kwi-CDNA MoLEKES nge-10 × bebardes kunye ne-UMI, ephantsi kolwakhiwo oluqhelekileyo lwethala leencwadi.

Iimbonakalo

● Ukulungiswa kokumiswa kwe-nuclei enye ye-nuclei ukusuka kwizicubu ze-frozen

● Ukuqulunqwa kwe-Gel Beads

● I-Bead nganye kwi-gem ilayishwe ngamacandelo ama-4:

I-Poly (i-DT) yomsila we-MRNA Priing kunye neCDNA Synthesis,

Isichazi esikhethekileyo semolekyuli (i-UMI) ukulungisa i-ames

I-10x barcode

Ukulandelelana kwenxalenye yokufunda inxenye enye

Izibonelelo

I-nucleus i-nucleus rna ye-nucleusing ijikeleza imida yendlela yokulandelelana kwe-RNA enye, eyenza:

● Ukusetyenziswa kweesampulu zeqhwa kwaye kungabi lula kwiisampulu ezintsha

● Uxinzelelo olusezantsi lweeseli eziqhwalelayo xa uthelekiswa nonyango lwe-enzymatic yeeseli ezintsha, eziboniswe kwidatha ye-transcriptome kwifom ye-genes eneengxaki ezincinci

● Akukho sidingo sokususwa kwangaphambi kokuba kwenziwe iiseli ezibomvu zegazi

● Ububanzi befowuni engenamda

● Uluhlu olukhulu lweesampulu ezifanelekileyo kuhlalutyo, kubandakanya iintlobo ezinzima nezibuthathaka ze-tissies ezinokuthathela ingqalelo kwiseli okanye intshabalalo ngexesha lokutshabalalisa izicubu

Iisampulu ezingenakwenziwa zihlalutye zihlalutyiwe yi-RNA ye-RNA kwaye zilungele ukulandelelana kwe-nuclei enye:

Iseli / izicubu	Isizathu
Phunga i-tishu yomkhenkce	Ayikwazi ukufumana imibutho emitsha okanye esele igcinwe
Iseli yemisipha, megakakorte, inqatha ...	I-selmeter yeseli inkulu kakhulu ukuba ingangenisi isixhobo
Isibindi ...	Ukonakala kakhulu ukwaphula, ukungakwazi ukwahlula iiseli enye
Iseli eNeuron, Ingqondo ...	Inovelwano ngakumbi, kulula ukugxininisa, iya kutshintsha iziphumo zokulandelelana
I-pancreas, i-hidroid ...	Sityebile kwi-endogenous endogenous, echaphazela imveliso yokumiswa kweseli enye

I-nucleus i-nucleus i-vs-iseli enye

I-nucleus enye	Iseli enye
Ububanzi befowuni engenamda	Ububanzi beSeli: 10-40 μm
Izixhobo zinokuba ziziculi ezinomkhenkce	Izinto ezizezinto kufuneka zibe ziziculi ezintsha
Uxinzelelo oluphantsi lweeseli eziqaqambileyo	Unyango lwe-enzyme lunokubangela uxinzelelo lweseli
Azikho iiseli ezibomvu zegazi kufuneka zisuswe	Iiseli ezibomvu zegazi kufuneka zisuswe
Inyukliya iveza i-biigulelo	Iseli iyonke ichaza i-biinorformation

Inkcazo

Iifoto zeSampula

Ithala leencwadi

Isicwangciso sokulandelelana

Idatha eyacetyiswayo

Ulawulo lwemeko

Izicubu zezilwanyana ≥ 200 mg

Izicubu zeziculi ≥ 400 mg

Ithala leencwadi le-10x ye-GEGNADS

I-Mulmina Pe150

I-100k ifundwe kwiseli nganye

(100-200 GB)

I-700-1200 i-Nuclei / μl kunye nokunyaniseka kwe-nuclei phantsi kwe-microscope

Ngeenkcukacha ezithe kratya kwiSikhokelo sokuLungiselela isampulu yesampulu kunye nokuHlala kweNkonzo, nceda uzive ukhululekile ukuthetha neIngcali ye-bmkgene