10x Genomics Visium Spatial Transcriptome
Likaroloana
● Qeto: 100 µM
● Spot Diameter: 55 µM
● Palo ea libaka: 4992
● Sebaka sa ho hapa: 6.5 x 6.5 mm
● Sebaka se seng le se seng se nang le barcode ho na le li-primers tse entsoeng ka likarolo tse 4:
- poly(dT) mohatla bakeng sa priming ea mRNA le cDNA synthesis
- Identifier e ikhethileng ea limolek'hule (UMI) ho lokisa leeme la ho holisa
- Barcode ea sebaka
- Tatelano e tlamang ea ho bala ha karolo e le 'ngoe ea tatellano
● H&E staining ea likarolo
Melemo
●Tšebeletso e le 'ngoe: e kopanya maitemogelo otlhe le dikgato tse di ikaegileng ka bokgoni, go akaretsa le cryo-sectioning, staining, tissue optimization, spatial barcoding, preparation laeborari, sequencing le bioinformatics .
● Sehlopha sa litsebi se nang le litsebo tse phahameng: o na le boiphihlelo mefuteng ea linama tse fetang 250 le mefuta e fetang 100+ ho kenyeletsoa batho, litoeba, liphoofolo tse anyesang, litlhapi le limela.
●Nchafatso ea nako ea 'nete morerong kaofela: ka taolo e felletseng ea tsoelopele ea liteko.
●Bioinformatics e akaretsang e tloaelehileng:sephutheloana se kenyelletsa litlhahlobo tse 29 le lipalo tse 100+ tsa boleng bo holimo.
●Tlhahlobo ea data le pono e ikhethileng: e fumaneha bakeng sa likopo tse fapaneng tsa lipatlisiso.
●Tlhahlobo e kopaneng ea boikhethelo ka tatellano ea sele e le 'ngoe ea mRNA
Litlhaloso
| Litlhoko tsa Mohlala | Laebrari | Leano la tatellano | Lintlha li khothalelitsoe | Taolo ea Boleng |
| Mehlala ea cryo e kentsoeng ka OCT, lisampole tsa FFPE (Bophara bo lekaneng: hoo e ka bang 6x6x6 mm3) Li-blocks tse 3 ka mohlala | Laeborari ea 10X Visium cDNA | Illumina PE150 | 50K PE e baloa sebakeng ka seng (60Gb) | RIN>7 |
Bakeng sa lintlha tse ling mabapi le tataiso ea boitokisetso ea mohlala le ts'ebetso ea ts'ebeletso, ka kopo ikutloe u lokolohile ho bua le aBMKGENE setsebi
Ts'ebetso ea ts'ebetso ea litšebeletso
Mokhahlelong oa boitokiso oa sampole, teko ea pele ea ho ntša RNA ka bongata e etsoa ho netefatsa hore RNA ea boleng bo holimo e ka fumanoa.Mokhahlelong oa ntlafatso ea lithane likarolo li silafatsoa le ho bonoa, 'me maemo a permeabilization bakeng sa tokollo ea mRNA ho tsoa liseleng a ntlafatsoa.Protocol e ntlafalitsoeng e sebelisoa nakong ea kaho ea laeborari, e lateloe ke tatellano le tlhahlobo ea data.
Ts'ebetso e felletseng ea ts'ebeletso e kenyelletsa lintlafatso tsa nako ea nnete le netefatso ea bareki ho boloka sephetho se arabelang, ho netefatsa ts'ebetso ea projeke e bonolo.
E kenyelletsa tlhahlobo e latelang:
Taolo ea Boleng ba Boitsebiso:
o Tlhahiso ea data le kabo ea lintlha tsa boleng
o Ho lemoha liphatsa tsa lefutso sebakeng ka seng
o Ho koaheloa ha lisele
Tlhahlobo e ka hare ea mohlala:
o Leruo la liphatsa tsa lefutso
o Ho kopanya matheba, ho kenyeletsoa tlhahlobo e fokolitsoeng ea boholo
o Tlhahlobo e fapaneng ea polelo lipakeng tsa lihlopha: ho tsebahatsa liphatsa tsa lefutso tsa matšoao
o Litlhaloso tse sebetsang le ho ntlafatsa liphatsa tsa lefutso tsa matšoao
Tlhahlobo ea lihlopha tse fapaneng
o Ho kopanngoa bocha ha matheba ho tsoa lisampoleng ka bobeli (mohlala, ho kula le ho laola) le ho kopanya hape
o Ho tsebahatsa liphatsa tsa lefutso tsa sehlopha ka seng
o Litlhaloso tse sebetsang le ho ntlafatsa liphatsa tsa lefutso tsa matšoao
o Phapang Polelo ea sehlopha se tšoanang lipakeng tsa lihlopha
Inner-sample Analysis
Spot clustering
Letshwao la liphatsa tsa lefutso le kabo ea sebaka
Inter-group Analysis
Motsoako oa data ho tsoa lihlopheng ka bobeli le ho kopanya hape
Liphatsa tsa lefutso tsa lihlopha tse ncha
Lekola tsoelopele e tsamaisoang ke BMKGene's spatial transcriptomics service ka 10X Visium Likhatisong tsena tse hlahang:
Chen, D. et al.(2023) 'mthl1, e ka bang Drosophila homologue ea mammalian adhesion GPCRs, e ameha liketsong tsa antitumor ho lisele tsa oncogenic tse entsoeng ka lintsintsi', Proceedings of the National Academy of Sciences of the United States of America, 120 (30), leq.e2303462120.doi: /10.1073/pnas.2303462120
Chen, Y. et al.(2023) 'STEEL e nolofalletsa tlhaloso e phahameng ea data ea transcriptomic ea spatiotemporal', Briefings in Bioinformatics, 24 (2), leqepheng la 1-10.doi: 10.1093/BIB/BBAD068.
Liu, C. et al.(2022) 'Atlas ea spatiotemporal ea organogenesis ho nts'etsopele ea lipalesa tsa orchid', Nucleic Acids Research, 50 (17), maq. 9724-9737.doi: 10.1093/NAR/GKAC773.
Wang, J. et al.(2023) 'Ho Kopanya Li-Spatial Transcriptomics le Motheo o le Mong oa RNA Sequencing e Senola Maqheka a Kalafo a Phekolo bakeng sa Uterine Leiomyoma', International Journal of Biological Sciences, 19 (8), maq. 2515-2530.doi: 10.7150/IJBS.83510.
