Full-Length mRNA Sequencing-Nanopore
Nta
● Kev ntes ntawm poly-A mRNA ua raws li cDNA synthesis thiab npaj tsev qiv ntawv
● Sequences ntawm cov ntawv sau tag nrho
● Bioinformatic tsom xam raws li kev ua kom haum rau ib qho kev siv genome
● Bioinformatic tsom xam suav nrog tsis tsuas yog qhia ntawm noob thiab isoform-theem tab sis kuj tsom xam ntawm lncRNA, noob fusions, poly-adenylation thiab noob qauv
Kev Pabcuam Zoo
●Quantification ntawm kev nthuav qhia ntawm qib isoform: ua kom muaj cov ncauj lus kom ntxaws thiab muaj tseeb, nthuav tawm qhov kev hloov pauv uas yuav raug npog thaum txheeb xyuas tag nrho cov kev qhia gene
●Txo cov ntaub ntawv thov:Piv rau Next-Generation Sequencing (NGS), Nanopore sequencing nthuav tawm cov ntaub ntawv qis dua, tso cai rau cov qib sib npaug ntawm cov noob qhia kom muaj nuj nqis saturation nrog cov ntaub ntawv me.
●Ntau qhov tseeb ntawm kev nthuav qhia kom muaj nuj nqis: ob qho tib si ntawm qib gene thiab isoform
●Kev txheeb xyuas cov ntaub ntawv transcriptomic ntxiv: lwm txoj kev polyadenylation, fusion noob thiab lcnRNA thiab lawv cov hom phiaj
●Kev txawj ntse: Peb pab neeg coj tau ntau yam kev paub dhau los rau txhua qhov haujlwm, tau ua tiav ntau dua 850 Nanopore cov ntawv sau ua haujlwm puv sijhawm thiab ua tiav ntau dua 8,000 tus qauv.
●Kev them nyiaj yug tom qab muag: Peb qhov kev cog lus txuas ntxiv dhau qhov kev ua tiav nrog rau 3 lub hlis tom qab muag kev pabcuam. Nyob rau lub sijhawm no, peb muab cov phiaj xwm taug qab, kev pab daws teeb meem, thiab Q&A ntu los daws cov lus nug ntsig txog cov txiaj ntsig.
Sample Requirements thiab Delivery
| Lub tsev qiv ntawv | Sequencing tswv yim | Cov ntaub ntawv pom zoo | Kev Tswj Xyuas Zoo |
| Poly A enriched | Illumina PE 150 | 6/12 Gb | Qhov qhab nia nruab nrab: Q10 |
Cov Qauv Xav Tau:
Nucleotides:
| Conc.(ng/μl) | Tus nqi (μg) | Purity | Kev ncaj ncees |
| ≥100 | ≥ 1.0 | OD 260/280 = 1.7-2.5 OD 260/230 = 0.5-2.5 Txwv los yog tsis muaj protein ntau lossis DNA kis tau pom ntawm gel. | Rau cov nroj tsuag: RIN≥7.0; Rau tsiaj: RIN≥7.5; 5.0 ≥28S / 18S≥1.0; txwv los yog tsis muaj qhov nce siab |
● Nroj tsuag:
Hauv paus, qia los yog Petal: 450 mg
Nplooj los yog noob: 300 mg
Txiv Hmab Txiv Ntoo: 1.2 g
● Tsiaj:
Lub plawv los yog txoj hnyuv: 300 mg
Viscera lossis hlwb: 240 mg
Cov leeg: 450 mg
Pob txha, plaub hau lossis tawv nqaij: 1g
● Arthropods:
qog: 6g
Crustacea: 300 mg
● Tag nrho cov ntshav:1 cev
● Cells: 106 hlwb
Pom zoo coj mus kuaj
Ntim: 2 ml centrifuge tube (Tin foil tsis pom zoo)
Sample labeling: Pab pawg + replicate xws li A1, A2, A3; B1, B2, B3.
Kev xa khoom:
1. Dry-ice: Cov qauv yuav tsum tau ntim rau hauv hnab thiab faus rau hauv cov dej khov qhuav.
2. RNAstable raj: RNA cov qauv tuaj yeem qhuav hauv RNA stabilization raj (xws li RNAstable®) thiab xa mus rau hauv chav tsev kub.
Kev Pabcuam Ua Haujlwm Flow
Nucleotides:
Qauv xa tuaj
Kev tsim kho tsev qiv ntawv
Sequencing
Cov ntaub ntawv tsom xam
Kev pab cuam tom qab muag
Kev Pabcuam Ua Haujlwm Flow
Cov ntaub so ntswg:
Kev sim tsim
Qauv xa tuaj
RNA rho tawm
Kev tsim kho tsev qiv ntawv
Sequencing
Cov ntaub ntawv tsom xam
Kev pab cuam tom qab muag
● Raw cov ntaub ntawv ua
● Kev txheeb xyuas cov ntaub ntawv
● Alternative splicing
● Kev nthuav qhia kom muaj nuj nqis hauv qib noob thiab qib isoform
● Kev txheeb xyuas qhov sib txawv
● Muaj nuj nqi annotation thiab txhawb ntxiv (DEGs thiab DETs)
Alternative splicing tsom xam
Alternative Polyadenylation Analysis (APA)
LncRNA kev twv ua ntej
Cov lus piav qhia ntawm cov noob caj noob ces
Clustering ntawm DETs
Protein-Protein Networks hauv DEGs
Tshawb nrhiav cov kev nce qib uas txhawb nqa los ntawm BMKGene's Nanopore tag nrho-ntev mRNA sequencing cov kev pabcuam los ntawm kev sau cov ntawv tshaj tawm.
Gong, B. et al. (2023) 'Epigenetic and transcriptional activation of the secretory kinase FAM20C as an oncogene in glioma', Journal of Genetics and Genomics, 50(6), pp. 422–433. doi: 10.1016/J.JGG.2023.01.008.
He, Z. et al. (2023) 'Full-length transcriptome sequencing of lymphocytes teb rau IFN-γ nthuav tawm Th1-skewed tiv thaiv kab mob hauv flounder (Paralichthys olivaceus),', Ntses & Shellfish Immunology, 134, p. 108636. doi: 10.1016/J.FSI.2023.108636.
Ma, Y. et al. (2023) 'Comparative analysis of PacBio and ONT RNA sequencing method for Nemopilema Nomurai venom identification', Genomics, 115(6), p. 110709. doi: 10.1016/J.YGENO.2023.110709.
Yus, D. et al. (2023) 'Nano-seq tsom xam qhia txog kev ua haujlwm sib txawv ntawm exosomes thiab microvesicles muab los ntawm hUMSC', Stem Cell Research thiab Therapy, 14(1), pp. 1–13. doi: 10.1186/S13287-023-03491-5/TABLES/6.
