● Kev ntes ntawm poly mRNA ua ntej npaj tsev qiv ntawv
● Kev ywj pheej ntawm ib qho kev siv genome: raws li de novo sib sau ua ke ntawm cov ntawv sau tseg, tsim cov npe ntawm cov unigenes uas tau sau tseg nrog ntau lub databases (NR, Swiss-Prot, COG, KOG, eggNOG, Pfam, GO, KEGG)
● Kev tshawb xyuas bioinformatic ntawm cov noob qhia thiab cov qauv sau ntawv
●Kev txawj ntse: nrog cov ntaub ntawv teev tseg ntawm kev ua tiav ntau dua 600,000 tus qauv ntawm BMKGENE, nthuav dav ntau hom qauv xws li kab lis kev cai ntawm tes, cov ntaub so ntswg, thiab cov kua dej hauv lub cev, peb pab pawg coj ntau yam kev paub rau txhua qhov haujlwm. Peb tau ua tiav kaw ntau dua 100,000 mRNA-Seq cov haujlwm hauv ntau qhov kev tshawb fawb.
●Rigorous Quality Control: peb siv cov ntsiab lus tseem ceeb hauv txhua theem, los ntawm cov qauv thiab kev npaj tsev qiv ntawv mus rau kev sib txuas lus thiab bioinformatics. Qhov kev soj ntsuam nruj me ntsis ua kom cov khoom xa tuaj raws li cov txiaj ntsig zoo.
● Cov lus piav qhia dav dav: peb siv ntau lub databases los ua haujlwm piav qhia qhov txawv ntawm Cov Genes (DEGs) thiab ua qhov kev txheeb xyuas qhov zoo sib xws, muab kev nkag siab ntawm cov txheej txheem cellular thiab molecular hauv qab cov lus teb.
●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.
Lub tsev qiv ntawv | Sequencing tswv yim | Cov ntaub ntawv pom zoo | Kev Tswj Xyuas Zoo |
Poly A enriched | Illumina PE 150 DNBSEQ-T7 | 6-10 Gb | Q30≥85% |
Nucleotides:
Conc.(ng/μl) | Tus nqi (μg) | Purity | Kev ncaj ncees |
≥ 10 | ≥ 0.2 | 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≥4.0; Rau tsiaj: RIN≥4.5; 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
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.
Bioinformatics
Transscriptome los ua ke thiab xaiv unigene
Unigene annotation
Kev sib raug zoo thiab kev ntsuam xyuas ntawm cov khoom siv roj ntsha
Differentially nthuav qhia Genes (DEGs)
Functional Annotation of DEGs
Functional Enrichment of DEGs
Tshawb nrhiav cov kev nce qib uas tau pab los ntawm BMKGene's eukaryotic NGS mRNA sequencing cov kev pabcuam los ntawm kev sau cov ntawv tshaj tawm.
Shen, F. et al. (2020) 'De novo transcriptome assembly and sex-biased gene expression in the gonads of Amur catfish (Silurus asotus)', Genomics, 112(3), pp. 2603–2614. doi: 10.1016/J.YGENO.2020.01.026.
Zhang C., et al. (2016) 'Transcriptome tsom xam ntawm sucrose metabolism thaum lub teeb o thiab kev loj hlob hauv dos (Allium cepa L.),', Frontiers in Plant Science, 7(September), p. 212763. doi: 10.3389/FPLS.2016.01425/BIBTEX.
Zhou, C. et al. (2017) 'De novo assembly, characterization and annotation for the transcriptome of Sarcoccheilichthys sinensis', PLoS ONE, 12(2). doi: 10.1371/JOURNAL.PONE.0171966.
Zou, L. et al. (2021) 'De novo transcriptome tsom xam muab kev nkag siab rau cov ntsev kam rau ntawm Podocarpus macrophyllus nyob rau hauv salinity stress', BMC Plant Biology, 21(1), pp. 1–17. doi: 10.1186/S12870-021-03274-1/FIGURES/9.