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I-BMKMANU S1000 Spatial Transcriptome

Isithombe Esifakiwe se-BMKMANU S1000 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 S1000 Spatial Transcriptome Chip, iziqhayisaukulungiswa okuthuthukisiweyokungu-5µM, ifinyelela kububanzi obuncane beselula, futhi inika amandlaizilungiselelo zokulungiswa kwamazinga amaningi. I-chip ye-S1000, equkethe cishe izindawo eziyizigidi ezingu-2, isebenzisa ama-microwell agqitshwe ngobuhlalu obulayishwe ama-probe wokuthwebula anebhakhodi yendawo. Umtapo wezincwadi we-cDNA, ohlanganiswe namabhakhodi wendawo, ulungiswa kusukela ku-chip ye-S1000 bese ulandelana ngokulandelana endaweni ye-Illumina NovaSeq. Inhlanganisela yamasampula anebhakhodi yendawo kanye nama-UMI kuqinisekisa ukunemba nokucaciswa kwedatha ekhiqizwayo. Isibaluli esiyingqayizivele se-chip ye-BMKManu S1000 silele ekusebenziseni kwayo izinto ezihlukahlukene, enikeza izilungiselelo zokuxazulula amaleveli amaningi ezingashunwa kahle kumathishu ahlukene namazinga emininingwane. Lokhu kuzivumelanisa nezimo kubeka i-chip njengenketho evelele yezifundo ezihlukahlukene ze-spatial transcriptomics, iqinisekisa ukuhlangana okunembayo kwendawo okunomsindo omncane.

Besebenzisa i-chip ye-BMKManu S1000 nobunye ubuchwepheshe be-transcriptomics yendawo, abacwaningi bangathola ukuqonda okungcono kwenhlangano yendawo yamangqamuzana kanye nokusebenzisana okuyinkimbinkimbi kwamangqamuzana okwenzeka ngaphakathi kwezicubu, kunikeze ukuqonda okubalulekile kwezinqubo eziyisisekelo zezinqubo zebhayoloji emikhakheni eminingi ehlukahlukene, kufaka phakathi. i-oncology, i-neuroscience, i-development biology, i-immunology kanye nezifundo ze-botanical.

I-Platform: BMKManu S1000 chip kanye ne-Illumina NovaSeq

Xhumana nathi

Imininingwane Yesevisi

I-Bioinformatics

Imiphumela yedemo

Okushicilelwe Okufakiwe

I-BMKMANU S1000 Spatial Transcriptome Technical Scheme

Izici

● Ukulungiswa: 5 µM

● Ububanzi be-Spot: 2.5 µM

● Inombolo yezindawo: cishe 2 Million

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

● Ubuhlalu obunebhakhodi ngabunye bulayishwa ama-primer akhiwe izigaba ezi-4:

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

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

Ibhakhodi yendawo

Ukulandelana okubophezelayo kokufunda okuyingxenye kokulandelana kwesiqalo esingu-1

● I-H&E ne-fluorescent staining yezigaba

● Amathuba okusebenzisaubuchwepheshe bokuhlukaniswa kwamaseli: ukuhlanganiswa kokungcoliswa kwe-H&E, ukufakwa kwamabala kwe-fluorescent, nokulandelana kwe-RNA ukuze kunqunywe imingcele yeseli ngalinye futhi kwabele isisho sofuzo ngendlela efanele kuseli ngalinye.

Izinzuzo ze-BMKMANU S1000

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

●Ukuhlaziywa Kwezixazululo Zezinga Eliningi:Ukuhlaziya okuguquguqukayo kwamazinga amaningi okusuka ku-100 μm kuya ku-5 μm ukuze kuxazululwe izici zezicubu ezihlukahlukene ngokulungiswa okufanele.

●Amathuba Okusebenzisa “Ezintathu kusilayidi esisodwa” Ubuchwepheshe Bengxenye Yeseli: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 nama-Multiple Sequencing Platforms: Kokubili i-NGS nokulandelana okufunde isikhathi eside kuyatholakala.

●Idizayini Eguquguqukayo Yendawo Yokuthwebula Esebenzayo engu-1-8: 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, ukubhala ikhodi yendawo, ukulungiswa komtapo wolwazi, 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 Ngokwezifiso kanye Nokubona: itholakalela izicelo zocwaningo ezahlukene

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

●Izibuyekezo zesikhathi sangempela kuphrojekthi yonke: ngokulawula okugcwele kokuqhubeka kokuhlolwa.

●Ukuhlaziya Okuhlangene Ongakukhetha Ngokulandelana kwe-mRNA yeseli elilodwa

Imininingwane Yesevisi

Isampula

Izimfuneko

Umtapowolwazi

Isu lokulandelanisa

Idatha iyanconywa

Ikhwalithi yokulawula

Amasampula e-cryo ashumekwe i-OCT, amabhulokhi angu-3 isampula ngayinye

Ilabhulali ye-cDNA ye-S1000

I-Illumina PE150 (ezinye izinkundla ziyatholakala)

100K PE ifundwa ngo-100 uM

( 60-150 Gb )

I-RIN>7

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

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.

Okwedlule: I-10x Genomics Visium Spatial Transcriptome

Olandelayo: Ukulandelana Kohlobo Lonke Lwezitshalo/Izilwane

Idatha ekhiqizwe i-BMKMANU S1000 ihlaziywa kusetshenziswa isofthiwe ye-“BSTMatrix”, eklanywe ngokuzimela yi-BMKGENE, ikhiqiza i-Gene Expression Matrix. Kusukela lapho, kukhiqizwa umbiko ojwayelekile ohlanganisa ukulawulwa kwekhwalithi yedatha, ukuhlaziywa kwesampula yangaphakathi kanye nokuhlaziywa kwamaqembu.

● Ukulawulwa Kwekhwalithi Yedatha:
Ukukhishwa kwedatha nokusabalalisa amaphuzu wekhwalithi
Ukutholwa kofuzo endaweni ngayinye
Ukumbozwa kwezicubu
● Ukuhlaziywa kwesampula yangaphakathi:
Ukunotha kofuzo
Ukuhlanganisa amabala, okuhlanganisa ukuhlaziywa kobukhulu obuncishisiwe
Ukuhlaziywa kwesisho esihlukile phakathi kwamaqoqo: ukuhlonza izakhi zofuzo zomaka
Isichasiselo esisebenzayo kanye nokunothiswa kwezakhi zofuzo zomaka
● Ukuhlaziya kwamaqembu:
Ukuphinda kuhlanganiswe amachashaza asuka womabili amasampuli (isb. okugulayo 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 okuthi “BSTViewer” iyithuluzi elisebenziseka kalula elenza umsebenzisi akwazi ukubona ngeso lengqondo ukusho kofuzo nokuhlanganisa amabala ezinqumweni ezihlukene.

I-BMKGene ithuthukise isoftware yokubuka ngeso okusebenziseka kalula

I-BSTViewer spot clustering ekulungisweni kwamazinga amaningi

I-BSTCelViewer: ukuhlukaniswa kwamaseli okuzenzakalelayo kanye nesandla

Ukuhlaziywa kwesampula yangaphakathi

Spot clustering:

Ukuhlonza izakhi zofuzo zikamaka nokusabalalisa kwendawo:

Inter-group Analysis

Inhlanganisela yedatha kusuka womabili amaqembu kanye neqoqo kabusha:

Izakhi zofuzo zomaka zamaqoqo amasha:

Hlola intuthuko egqugquzelwa izinsiza ze-BMKGene zokubhaliwe ngobuchwepheshe be-BMKManu S1000 kulokhu kushicilelwa okufakiwe:

Ingoma, X. et al. (2023) 'I-Spatial transcriptomics iveza amaseli e-chlorenchyma enziwe ukukhanya abandakanyeka ekukhuthazeni ukuvuselelwa kwehlumela ku-tomato callus',Izinqubo zeNational Academy of Sciences yase-United States of America, 120(38), p. e2310163120. doi: 10.1073/pnas.2310163120

Wena, Y. et al. (2023) 'Ukuqhathaniswa kwesistimu yezindlela zokubhalwa kwezindawo ezisekelwe ngokulandelana',i-bioRxiv, p. 2023.12.03.569744. doi: 10.1101/2023.12.03.569744.