Single-nuclei RNA Sequencing
Sekema sa Tekheniki
Ho aroloa ha li-nuclei ho finyelloa ke 10 × Genomics Chromium™, e nang le tsamaiso ea microfluidics e nang le mechine e robeli e nang le ho tšela habeli. Tsamaisong ena, lifaha tsa gel tse nang le li-barcode le primer, li-enzyme le nucleus e le 'ngoe li kenngoa ka lerotholi la oli ea boholo ba nanoliter, e hlahisang Gel Bead-in-Emulsion (GEM). Hang ha GEM e thehoa, lisele tsa lysis le ho lokolloa ha barcode li etsoa ho GEM ka 'ngoe. mRNA e fetisetsoa ka morao ho limolek'hule tsa cDNA tse nang le 10 × barcodes le UMI, tse tlas'a taolo ea kaho e tloaelehileng ea tatellano ea laeborari.
Likaroloana
● Tokiso ea ho emisoa ha li-nuclei tse le 'ngoe ho tloha liseleng tse leqhoa
● Ho thehoa ha Gel Bead-in-Emulsion (GEM) e lateloa ke cDNA synthesis
● Sefaha se seng le se seng ho GEM se na le li-primers tse entsoeng ka likarolo tse 4:
poly(dT) mohatla bakeng sa priming ea mRNA le cDNA synthesis,
Unique Molecular Identifier (UMI) ho lokisa leeme la ho holisa
10x barcode
Tatelano e tlamang ya karolo e itseng e baletsweng 1 ya tatellano
Melemo
Tatelano ea "nucleus" e le 'ngoe ea RNA e qoba meeli ea tatellano ea sele e le 'ngoe ea RNA, e nolofalletsang:
● Tšebeliso ea lisampole tse hatselitsoeng eseng feela lisampole tse ncha
● Khatello e tlase ea lisele tse hoamisitsoeng ha e bapisoa le kalafo ea enzymatic ea lisele tse foreshe, e bonts'ang data ea transcriptome ka mofuta oa liphatsa tsa lefutso tse sa bakeng khatello ea maikutlo.
● Ha ho hlokahale hore lisele tse khubelu tsa mali li tlosoe pele
● Sekhahla se se nang moeli sa sele
● Mehlala e mengata e loketseng hore e hlahlojoe, ho akarelletsa le lisele tse rarahaneng le tse robehang habonolo tse atisang ho hohla kapa ho senyeha ha lisele li arohana.
Mehlala e ke keng ea hlahlojoa ka tatellano ea sele e le 'ngoe ea RNA mme e lokeloa ke tatellano ea li-nuclei tse le 'ngoe tsa RNA:
| Sele/ Sehlaha | Lebaka |
| Lithipa tse sa hloekang tse hoammeng | Ha e khone ho fumana mekhatlo e mecha kapa e bolokiloeng nako e telele |
| Lisele tsa mesifa, Megakaryocyte, Mafura ... | Bophara ba sele bo boholo haholo hore bo ka kena ka har'a sesebelisoa |
| Sebete... | E robeha haholo hore e ka robeha, ha e khone ho khetholla lisele tse le 'ngoe |
| Neuron cell, boko… | Ho utloisisa haholoanyane, ho bonolo ho hatella, ho tla fetola liphetho tsa tatellano |
| Makhopho, Thyroid ... | E ruile ka li-enzyme tsa endogenous, tse amang tlhahiso ea ho emisoa ha sele e le 'ngoe |
Nucleus e le 'ngoe vs Lesele e le 'ngoe
| Nucleus e le 'ngoe | Sele e le 'ngoe |
| Sekhahla se se nang moeli sa sele | Bophara ba sele: 10-40 μm |
| Thepa e ka ba lesela le leqhoa | Boitsebiso bo tlameha ho ba lisele tse ncha |
| Khatello e tlase ea lisele tse leqhoa | Kalafo ea enzyme e ka baka karabelo ea khatello ea kelello |
| Ha ho na lisele tse khubelu tsa mali tse lokelang ho tlosoa | Lisele tse khubelu tsa mali li hloka ho tlosoa |
| Nuclear e hlahisa bioinformation | Sele eohle e hlahisa litaba tsa bophelo |
Litlhaloso
| Litlhoko tsa Mohlala | Laebrari | Leano la tatellano | Lintlha li khothalelitsoe | Taolo ea Boleng |
| Lithipa tsa liphoofolo ≥ 200 mg Semela sa semela ≥ 400 mg | 10x Genomics sn cDNA laebrari | Illumina PE150 | 100K PE e baloa ka sele (100-200 Gb) | 700-1200 nuclei/μl le nuclei botšepehi bo bonoa tlas'a microscope |
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
Phallo ea Mosebetsi oa Ts'ebeletso
E kenyelletsa tlhahlobo e latelang:
● Taolo ea Boleng: palo ea lisele, ho lemoha liphatsa tsa lefutso, boitsebiso bo nepahetseng ba lisele, limolek'hule tsa RNA le tlhaloso ea mantsoe.
● Tlhahlobo ea Mehlala e ka Hare:
Khokahano ea lisele le tlhaloso ea sehlopha
Differential Expression Analysis: ho khetholla li-DEG ka lihlopha
Litlhaloso tse sebetsang le ntlafatso ea li-DEG tsa lihlopha
● Tlhahlobo ea lihlopha tse fapaneng:
Motsoako oa data
Differential Expression Analysis: ho khetholla li-DEG ka lihlopha
Tlhaloso ea ts'ebetso le ntlafatso ea lihlopha tsa DEGs
● Tlhahlobo e tsoetseng pele:
Tlhahlobo ea potoloho ea lisele
Tlhahlobo ea pseudotime
Tlhahlobo ea puisano ea mehala (CellPhoneDB)
Gene Set Enrichment Analysis (GSEA)
Tlhahlobo e ka hare ea mohlala
Khokahano ea lisele:
Phapang ea Tlhaloso ea Polelo: Cluster DEGs
Tlhahlobo ea lihlopha tse fapaneng
Tlhaloso e fapaneng ea Polelo: Lihlopha tsa DEGs
Tlhahlobo e tsoetseng pele:
Tlhahlobo ea pseudotime:
Tlhahlobo ea potoloho ea lisele:
Lekola tsoelo-pele e tsamaisoang ke BMKGene's single-nucleus RNA tatellano ea litšebeletso ka 10X Chromium likhatisong tsena tse hlahang:
Wang, L. et al. (2021) 'Tlhahlobo ea transcriptomic ea sele e le' ngoe e senola boemo ba 'mele ba ho itšireletsa mafung ka ho ata ha asthma e hanyetsanang le steroid',Melaetsa ea National Academy of Sciences ea United States of America, 118(2), leq. e2005590118. doi: 10.1073/pnas.2005590118
Zheng, H. et al. (2022) 'Global Regulatory Network for Dysregulated Gene Expression and Abnormal Metabolic Signaling in Immune Cells in the Microenvironment of Graves' Disease le Hashimoto's Thyroiditis',Frontiers ho Immunology, 13, leq. 879824. doi: 10.3389/FIMMU.2022.879824/BIBTEX.
Tian, H. et al. (2023) 'Single-cell transcriptome e senola heterogeneity le likarabo tsa' mele tsa 'mele tsa leukocyte ka mor'a ho entoa ka Edwardsiella tarda e sa sebetseng ka flounder (Paralichthys olivaceus)',Tlhapi ea metsing, 566, leq. 739238. doi: 10.1016/J.AQUACULTURE.2023.739238.
Yu, Y. le ba bang. (2023) 'Phekolo ea Photodynamic e ntlafatsa sephetho sa li-immune checkpoint inhibitors ka ho nchafatsa boits'ireletso ba anti-tumor ho bakuli ba mofets'e oa mpa',Kankere ea Gastric, 26(5), leqepheng la 798-813. doi: 10.1007/S10120-023-01409-X/METRICS.
