Tib- nuclei RNA Sequencing
Tswv yim
Kev sib cais ntawm nuclei yog ua tiav los ntawm 10 × Genomics Chromium ™, uas muaj yim-channel microfluidics system nrog ob txoj kev hla. Nyob rau hauv no system, ib tug gel hlaws dai nrog barcodes thiab primer, enzymes thiab ib tug tib nucleus yog encapsulated nyob rau hauv nanoliter-loj roj poob, generating Gel Bead-in-Emulsion (GEM). Thaum GEM raug tsim, cell lysis thiab tso tawm barcodes tau ua nyob rau hauv txhua GEM. mRNA yog thim rov qab rau hauv cDNA molecules nrog 10 × barcodes thiab UMI, uas yog ntxiv rau cov qauv sib txuas hauv tsev qiv ntawv.
Nta
● Kev npaj ntawm ib leeg-nuclei raug tshem tawm los ntawm cov ntaub so ntswg khov
● Tsim cov Gel Bead-in-Emulsion (GEM) ua raws li cDNA synthesis
● Txhua hlaws hauv ib lub GEM yog loaded nrog primers muaj li ntawm 4 seem:
poly (dT) tail rau mRNA priming thiab cDNA synthesis,
Unique Molecular Identifier (UMI) los kho qhov kev hloov pauv tsis zoo
10 x barcode
Binding sequence ntawm ib feem nyeem 1 sequencing primer
Qhov zoo
Single-nucleus RNA sequencing circumvents cov kev txwv ntawm ib leeg-cell RNA sequencing, ua rau:
● Kev siv cov qauv khov thiab tsis txwv rau cov qauv tshiab
● Tsis tshua muaj kev ntxhov siab ntawm cov hlwb khov thaum piv rau kev kho enzymatic ntawm cov hlwb tshiab, muaj kev cuam tshuam hauv cov ntaub ntawv transcriptome nyob rau hauv daim ntawv ntawm cov noob muaj kev ntxhov siab tsawg.
● Tsis xav tau kev tshem tawm cov qe ntshav liab ua ntej
● Unlimited cell txoj kab uas hla
● Cov qauv loj ntawm cov qauv tsim nyog rau kev tshuaj xyuas, suav nrog cov ntaub so ntswg nyuaj thiab tsis yooj yim uas ua rau cov cell clumping lossis kev puas tsuaj thaum cov ntaub so ntswg dissociation
Cov qauv uas tsis tuaj yeem txheeb xyuas los ntawm ib leeg-cell RNA sequencing thiab tsim nyog rau ib qho nuclei RNA sequencing:
| Cell / daim ntaub | Yog vim li cas |
| Unfresh khov cov ntaub so ntswg | Tsis tuaj yeem tau txais cov koom haum tshiab lossis kev txuag ntev |
| Muscle cell, Megakaryocyte, Fat… | Cell txoj kab uas hla yog loj dhau mus rau hauv lub ntsuas |
| Lub siab… | Yooj yim heev rau tawg, tsis tuaj yeem paub qhov txawv ntawm ib leeg |
| Neuron cell, Lub hlwb… | Ntau rhiab heev, yooj yim rau kev ntxhov siab, yuav hloov cov txiaj ntsig sib luag |
| Pancreas, thyroid… | nplua nuj nyob rau hauv endogenous enzymes, cuam tshuam rau zus tau tej cov cell suspension |
Single-nucleus vs Single-cell
| Ib leeg-nucleus | Ib leeg xwb |
| Unlimited cell diam | Cell txoj kab uas hla: 10-40 μm |
| Cov khoom tuaj yeem yog cov ntaub so ntswg khov | Cov khoom yuav tsum yog cov ntaub so ntswg tshiab |
| Tsawg kev ntxhov siab ntawm cov cell khov | Kev kho enzyme tuaj yeem ua rau muaj kev ntxhov siab ntawm tes |
| Tsis muaj cov qe ntshav liab yuav tsum tau muab tshem tawm | Cov qe ntshav liab yuav tsum tau muab tshem tawm |
| Nuclear qhia txog bioinformation | Tag nrho cov cell qhia txog bioinformation |
Specifications
| Cov Qauv Xav Tau | Lub tsev qiv ntawv | Sequencing tswv yim | Cov ntaub ntawv pom zoo | Kev Tswj Xyuas Zoo |
| Tsiaj cov ntaub so ntswg ≥ 200 mg Cov ntaub so ntswg ≥ 400 mg | 10x Genomics sn cDNA tsev qiv ntawv | Illumina PE 150 | 100K PE nyeem ib lub xov tooj (100-200 Gb) | 700-1200 nuclei / μl thiab nuclei kev ntseeg tau pom nyob rau hauv lub tshuab tsom |
Yog xav paub ntxiv txog cov qauv qhia kev npaj thiab kev pabcuam kev ua haujlwm, thov koj tham nrog aBMKGENE tus kws tshaj lij
Kev Pabcuam Ua Haujlwm Flow
Xws li cov kev txheeb xyuas hauv qab no:
● Kev Tswj Xyuas Zoo: pes tsawg lub hlwb, kuaj cov noob, txheeb xyuas cov hlwb kom raug, RNA molecules thiab kev qhia kom muaj nuj nqis
● Inner Sample Analysis:
Cell clustering thiab cluster annotation
Differential Expression Analysis: Kev txheeb xyuas ntawm DEGs hauv pawg
Cov lus piav qhia ua haujlwm thiab ua kom muaj txiaj ntsig ntawm pawg DEGs
● Kev Ntsuam Xyuas Cov Pab Pawg Sib Koom:
Kev sib xyaw ntawm cov ntaub ntawv
Differential Expression Analysis: Kev txheeb xyuas ntawm DEGs hauv pawg
Cov lus piav qhia ua haujlwm thiab txhawb nqa pawg DEGs
● Advanced Analysis:
Kev txheeb xyuas lub voj voog ntawm tes
Pseudotime tsom xam
Kev soj ntsuam kev sib txuas lus ntawm tes (CellPhoneDB)
Gene Set Enrichment Analysis (GSEA)
Sab hauv qauv tsom xam
Cell clustering:
Differential Expression Analysis: pawg DEGs
Inter-pab pawg tsom xam
Differential Expression Analysis: Pawg DEGs
Advanced Analysis:
Pseudotime tsom xam:
Cell Cycle Analysis:
Tshawb nrhiav cov kev nce qib tau yooj yim los ntawm BMKGene cov kev pabcuam ib leeg-nucleus RNA sequencing los ntawm 10X Chromium hauv cov ntawv tshaj tawm no:
Wang, L. et al. (2021) 'Ib leeg-cell transcriptomic tsom xam qhia lub cev tiv thaiv kab mob ntawm lub ntsws hauv steroid-resistant hawb pob exacerbation',Cov txheej txheem ntawm National Academy of Sciences ntawm Tebchaws Meskas, 118(2), p. e2005590118. doi: 10.1073/pnas.2005590118
Zheng H., et al. (2022) 'Lub Ntiaj Teb Kev Tswj Xyuas Network rau Dysregulated Gene Expression thiab Abnormal Metabolic Signaling in Immune Cells in Microenvironment of Graves' Disease thiab Hashimoto's Thyroiditis',Frontiers hauv Immunology, 13, ib. 879824. doi: 10.3389/FIMMU.2022.879824/BIBTEX.
Tian, H. et al. (2023) 'Single-cell transcriptome uncovers heterogeneity and immune responses of leukocytes tom qab txhaj tshuaj tiv thaiv kab mob Edwardsiella tarda hauv flounder (Paralichthys olivaceus)',AquacultureIb., 566, p. 739238. doi: 10.1016/J.AQUACULTURE.2023.739238.
Yus, Y. et al. (2023) 'Photodynamic therapy txhim kho cov txiaj ntsig ntawm kev tiv thaiv kab mob tiv thaiv kab mob los ntawm kev kho cov qog nqaij hlav tiv thaiv kab mob hauv cov neeg mob qog noj ntshav',Mob plab, 26(5), pp. 798–813. doi: 10.1007/S10120-023-01409-X/METRICS.
