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条形 isibhengezo-03

Imikhiqizo

I-BMKMANU S3000_Spatial Transcriptome

I-Spatial transcriptomics imi phambili ekusungulweni kwesayensi, inika amandla abacwaningi ukuthi bahlole amaphethini enkulumo eyinkimbinkimbi yezakhi zofuzo phakathi kwezicubu kuyilapho belondoloza isimo sabo sendawo. Phakathi kwamapulatifomu ahlukahlukene, i-BMKGene ithuthukise i-BMKManu S3000 Spatial Transcriptome Chip, eziqhayisa ngokulungiswa okuthuthukisiwe kwe-3.5µm, efinyelela ububanzi obuncane, futhi ivumela izilungiselelo zokuxazulula amazinga amaningi. I-chip ye-S3000, equkethe cishe izindawo eziyizigidi ezi-4, isebenzisa ama-microwell agqitshwe ngobuhlalu obulayishwe ama-probe wokuthwebula anebhakhodi. Umtapo wezincwadi we-cDNA, ohlanganiswe namabhakhodi wendawo, ulungiswa kusukela ku-chip ye-S3000 bese ulandelana ngokulandelana endaweni ye-Illumina NovaSeq. Inhlanganisela yamasampula anebhakhodi yendawo kanye nama-UMI kuqinisekisa ukunemba nokucaciswa kwedatha ekhiqizwayo. I-chip ye-BMKManu S3000 iguquguquka kakhulu, inikezela ngezilungiselelo zokuxazulula amazinga amaningi ezingashunwa kahle kumathishu ahlukene kanye namazinga afiswayo emininingwane. Lokhu kuzivumelanisa nezimo kubeka i-chip njengenketho evelele yezifundo ezihlukahlukene ze-spatial transcriptomics, iqinisekisa ukuhlangana okunembayo kwendawo okunomsindo omncane. Ukusetshenziswa kobuchwepheshe bokuhlukanisa amaseli nge-BMKManu S3000 kuvumela ukunqunywa kwedatha yokubhala emikhawulweni yamaseli, okuholela ekuhlaziyweni okunencazelo eqondile yebhayoloji. Ngaphezu kwalokho, ukulungiswa okuthuthukisiwe kwe-S3000 kuphumela enanini eliphezulu lezakhi zofuzo nama-UMI atholiwe ngeseli ngalinye, okuvumela ukuhlaziya okunembe kakhulu kwamaphethini okulotshwa kwendawo nokuhlanganisa amaseli.


Imininingwane Yesevisi

I-Bioinformatics

Imiphumela yedemo

Umehluko phakathi kwe-S3000 ne-S1000

I-BMKMANU S3000 Spatial Transcriptome Technical Scheme

未标题-1-01(1)

Izici

- Ukulungiswa: 3.5 µM

- Ububanzi bendawo: 2.5 µM

- Inombolo yezindawo: cishe 4 Million

- 3 amafomethi angenzeka wendawo yokuthwebula: 6.8 mm * 6.8 mm, 11 mm * 11 mm noma 15 mm * 20 mm

- Ubuhlalu obunebhakhodi ngamunye bulayishwa ama-primers akhiwe izigaba ezi-4:

• umsila we-poly(dT) we-mRNA priming kanye ne-cDNA synthesis,

• Isihlonzi se-Molecular (UMI) ukuze silungise ukuchema kokukhulisa

• Ibhakhodi yendawo

• Ukulandelana okubophezelayo kokufunda ingxenye engu-1 yokuqalisa ngokulandelanayo

- H&E kanye ne-fluorescent staining yezigaba

- Amathuba okusebenzisa ubuchwepheshe bokuhlukaniswa kwamaseli: ukuhlanganiswa kokungcoliswa kwe-H&E, ukungcoliswa kwe-fluorescent, nokulandelana kwe-RNA ukuze kunqunywe imingcele yeseli ngalinye futhi kwabele ngokufanelekile ukubonakaliswa kofuzo kuseli ngalinye. Icubungula ukuhlaziywa kwephrofayela ye-Spatial engezansi komfula okusekelwe ku-cell bin.

- Kungenzeka ukuzuza ukuhlaziywa kwesinqumo sezinga eliphezulu: Ukuhlaziya okuguquguqukayo kwamazinga amaningi kusukela ku-100um kuya ku-3.5 um ukuxazulula izici zezicubu ezihlukahlukene ngokulungiswa okuphelele.

Izinzuzo ze-BMKMANU S3000

-Ukuphindwa kabili kwezindawo zokuthwebula kufika ku-4 Million: ngokulungiswa okuthuthukisiwe kwe-3.5 uM, okuholela ekutholeni isakhi sofuzo esiphezulu kanye ne-UMI ngeseli ngayinye. Lokhu kuphumela ekuhlanganisweni okuthuthukisiwe kwamaseli ngokusekelwe kumaphrofayili abhaliwe, anemininingwane emincane efana nesakhiwo sezicubu.

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- Ukulungiswa kwamaselula angaphansi:Indawo ngayinye yokuthwebula iqukethe > Izindawo EzineBarcode eziyizigidi ezingu->2 ezinobubanzi obungu-2.5 µm kanye nesikhala esingu-5 µm phakathi kwezindawo zamabala, okuvumela ukuhlaziywa kwe-spatical transcriptome ngokulungiswa kwamaselula amancane (5 µm).

-Ukuhlaziya ukulungiswa kwamaleveli amaningi:Ukuhlaziya okuguquguqukayo kwamazinga amaningi okusuka ku-100 μm kuya ku-5 μm ukuze kuxazululwe izici zezicubu ezihlukahlukene ngokulungiswa okufanele.

-Amathuba okusebenzisa ubuchwepheshe bokuhlukanisa amaseli "Amathathu kusilayidi esisodwa":ukuhlanganisa amabala e-fluorescence, ukugcotshwa kwe-H&E, nokulandelana kwe-RNA kusilayidi esisodwa, i-algorithm yethu yokuhlaziya "ezintathu kokukodwa" inika amandla ukukhonjwa kwemingcele yeseli yama-transcriptomics asuselwa kumaseli alandelayo.

-Ihambisana nezinkundla eziningi zokulandelana: kokubili i-NGS nokulandelana okufunde isikhathi eside kuyatholakala.

-Idizayini eguqukayo yendawo engu-1-8 yokuthwebula esebenzayo: usayizi wendawo yokuthwebula uyavumelana nezimo, ukwazi ukusebenzisa amafomethi angu-3 (6.8 mm * 6.8 mm., 11 mm * 11 mm kanye no-15 mm * 20 mm)

-Isevisi yokuma okukodwa: ihlanganisa konke okuhlangenwe nakho nezinyathelo ezisuselwe emakhono, okuhlanganisa ukuhlukanisa i-cryo, ukungcolisa amabala, ukwenziwa ngcono kwezicubu, ukufaka ibhakhodi yendawo, ukulungiswa komtapo wezincwadi, ukulandelana, kanye ne-bioinformatics.

-I-bioinformatics ephelele kanye nokubonwa kwemiphumela okusebenziseka kalula:iphakheji ihlanganisa ukuhlaziya okungu-29 kanye nezibalo ezingu-100+ zekhwalithi ephezulu, kuhlanganiswe nokusetshenziswa kwesofthiwe ethuthukisiwe yangaphakathi ukuze ubone ngeso lengqondo nokwenza ngendlela oyifisayo ukuhlukaniswa kwamaseli nokuhlanganisa amabala.

-Ukuhlaziywa kwedatha okwenziwe ngendlela oyifisayo nokubona ngeso lengqondo: itholakalela izicelo zocwaningo ezahlukene

-Ithimba lobuchwepheshe elinamakhono aphezulu: onolwazi ngezinhlobo zezicubu ezingaphezu kuka-250 nezinhlobo eziyi-100+ ezihlanganisa abantu, igundane, izilwane ezincelisayo, izinhlanzi nezitshalo.

-Izibuyekezo zesikhathi sangempela kuyo yonke iphrojekthi: ngokulawula okugcwele kokuqhubeka kokuhlolwa.

-Ukuhlaziya okuhlangene ongakukhetha nokulandelana kwe-Single-cell mRNA

Imininingwane Yesevisi

Izidingo Zesampula Umtapowolwazi Isu Lokulandelanisa Idatha iyanconywa Ikhwalithi yokulawula

Amasampula e-cryo ashumekwe OCT

(Ubukhulu obuphelele: approx.

6×6×6 mm³)

2 amabhlogo isampula ngayinye

1 okokuhlola, 1 okokwenza isipele

I-S3000 cDNA Library I-Ilumina PE150 160K PE ifundeka ngo-100υM (250 Gb) I-RIN > 7

Ukuze uthole imininingwane eyengeziwe ngomhlahlandlela wokulungiselela isampula kanye nokuhamba komsebenzi wesevisi, sicela ukhululeke ukukhuluma no-a

Ukugeleza Komsebenzi Wesevisi

Esigabeni sokulungiselela isampula, uhlolo lokuqala lwenqwaba ye-RNA lwenziwa ukuze kuqinisekiswe ukuthi i-RNA yekhwalithi ephezulu ingatholakala. Esigabeni sokuthuthukisa izicubu izigaba ziba namabala futhi zibonwa ngeso lengqondo futhi nezimo ze-permeabilization zokukhululwa kwe-mRNA ezicutshini ziyathuthukiswa. Iphrothokholi elungiselelwe ibe isisetshenziswa phakathi nokwakhiwa komtapo wolwazi, kulandelwa ukulandelana nokuhlaziya idatha.

Ukugeleza komsebenzi okuphelele kwesevisi kuhilela izibuyekezo zesikhathi sangempela neziqinisekiso zeklayenti ukuze kugcinwe iluphu yempendulo ephendulayo, okuqinisekisa ukwenziwa kwephrojekthi okushelelayo.

 

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  • Okwedlule:
  • Olandelayo:

  • i-asd (1)

    Idatha ekhiqizwe yi-BMKMANU S3000 ihlaziywa kusetshenziswa isofthiwe ye-“BSTMatrix”, eklanywe ngokuzimela yi-BMKGENE, ekhiqiza ileveli yeseli kanye nesixazululo se-Gene Expression Matrix ngamaleveli amaningi. Kusukela lapho, kukhiqizwa umbiko ojwayelekile ohlanganisa ukulawulwa kwekhwalithi yedatha, ukuhlaziywa kwesampula yangaphakathi kanye nokuhlaziywa kwamaqembu.

    - Ukulawulwa Kwekhwalithi Yedatha:

    - Okukhipha idatha kanye nokusatshalaliswa kwekhwalithi yekhwalithi

    - Ukutholwa kofuzo endaweni ngayinye

    - Ukumbozwa kwezicubu

    - Ukuhlaziywa kwesampula kwangaphakathi:

    - Ukunotha kofuzo

    - Ukuhlanganisa amabala, okuhlanganisa ukuhlaziywa kobukhulu obuncishisiwe

    - Ukuhlaziywa kwesisho esihlukile phakathi kwamaqoqo: ukuhlonza izakhi zofuzo zomaka

    - Isichasiselo esisebenzayo kanye nokunothiswa kwezakhi zofuzo zomaka

    - Inter-group analysis

    - Ukuhlanganiswa kabusha kwamachashaza kuwo womabili amasampula (isb. aphethwe yisifo nokulawula) kanye nokuhlanganisa kabusha

    - Ukuhlonza izakhi zofuzo zomaka zeqoqo ngalinye

    - Isichasiselo esisebenzayo kanye nokunothiswa kwezakhi zofuzo zomaka

    - Ukuvezwa Okuhlukile kweqoqo elifanayo phakathi kwamaqembu

    Ukwengeza, i-BMKGene ithuthukiswe i-“BSTViewer” iyithuluzi elisebenziseka kalula elenza umsebenzisi akwazi ukubona ngeso lengqondo ukusho kofuzo nokuhlanganisa amabala ezinqumweni ezihlukene.

    i-asd (2)

    i-asd (3)

     

    I-BMKGene inikezela ngezinsizakalo zokuhlonza indawo ngokulungiswa kweseli elilodwa okunembile (ngokusekelwe kubhini weseli noma umgqomo wesikwele wamaleveli amaningi ukusuka ku-100um ukuya ku-3.5um).

     

    Idatha yokuhlonza indawo evela ezigabeni zethishu kusilayidi se-S3000 yenze kahle kanjena ngezansi.

    Indaba 1: Ubuchopho begundane

    xv (1)

    Ukuhlaziywa kwesigaba sobuchopho begundane nge-S3000 kuholele ekuhlonzweni kwamaseli angu-~94 000, ngokulandelana okumaphakathi kwezakhi zofuzo ezingu-~2000 ngeseli ngayinye. Ukulungiswa okuthuthukisiwe kwe-3.5 uM kubangele ukuhlanganisa okuningiliziwe kwamaseli okusekelwe emaphethini okuloba, namaqoqo amaseli alingisa izakhiwo ezihlukene zobuchopho. Lokhu kubonakala kalula ngokubona ngeso lengqondo ukusatshalaliswa kwamaseli ahlanganiswe njengama-oligodendrocyte namaseli e-microglia, acishe abekwe ngokukhethekile endaweni empunga nemhlophe, ngokulandelana.

     

    xv (1)

    Isifundo 2: Umbungu wegundane

    xv (1)

    Ukuhlaziywa kwesigaba sombungu wegundane nge-S3000 kuholele ekuhlonzweni kwamaseli angu-~2200 000, nokulandelana okumaphakathi kwezakhi zofuzo ezingu-~1600 ngeseli ngayinye. Ukulungiswa okuthuthukisiwe kwe-3.5 uM kubangele ukuhlanganisa okuningiliziwe kwamaseli okusekelwe emaphethini okuloba, namaqoqo angu-12 endaweni yeso kanye namaqoqo angu-28 endaweni yobuchopho.

    xv (1)

    Iqoqo leseli lokuhlaziya isampula yangaphakathi:

    xv (1)

    Ukuhlonza izakhi zofuzo zikamaka nokusabalalisa kwendawo:

    xv (1)

    - ukulungiswa okuphezulu kwamaselula angaphansi: Uma kuqhathaniswa nesilayidi se-S1000, indawo ngayinye yokuthwebula engu-S3000 iqukethe > Izindawo Ezinebhakhodi eziyizigidi ezingu-4 ezinobubanzi obungu-2.5 µm nebanga elingu-3.5 µm phakathi kwezikhungo, okuvumela ukuhlaziywa kwe-transcriptome yendawo ngokulungiswa kwamaselula aphezulu (umgqomo wesikwele: 3.5 µm).

    - Ukusebenza kahle kokuthwebula okuphezulu: uma kuqhathaniswa nesilayidi se-S1000, i-Median_UMI ikhuphuka isuka ku-30% iye ku-70%, i-Median_Gene ikhuphuka isuka ku-30% iye ku-60%

    Uhlelo lwe-chip ye-S1000:

    i-asd (1)

    Uhlelo lwe-chip ye-S3000:

    i-asd (2)

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