Ntev tsis-coding sequencing-Illumina
Kev Pabcuam Zoo
● Kev Pabcuam Zoo
● Cellular thiab cov ntaub so ntswg tshwj xeeb
● Cov theem tshwj xeeb qhia thiab nthuav qhia kev hloov pauv hloov pauv
● Cov qauv meej ntawm lub sijhawm thiab qhov chaw qhia
● Kev tsom xam nrog mRNA cov ntaub ntawv.
● BMKCloud-raws li kev xa tawm: Cov ntaub ntawv kho kom haum-tsim muaj nyob rau ntawm lub platform.
● Cov kev pab cuam tom qab muag siv tau rau 3 lub hlis tom qab qhov project tiav
Sample Requirements thiab Delivery
| Lub tsev qiv ntawv | Platform | Cov ntaub ntawv pom zoo | Cov ntaub ntawv QC |
| rRNA depletion | Illumina PE 150 | 10 Gb | Q30≥85% |
| Conc. (ng / μl) | Tus nqi (μg) | Purity | Kev ncaj ncees |
| ≥100 | ≥ 0.5 | OD 260/280 = 1.7-2.5 OD 260/230 = 0.5-2.5 Txwv tsis pub lossis tsis muaj protein ntau lossis DNA kis tau pom ntawm gel. | Rau cov nroj tsuag: RIN≥6.5; Rau tsiaj: RIN≥7.0; 5.0 ≥28S / 18S≥1.0; txwv los yog tsis muaj qhov nce siab |
Nucleotides:
Cov ntaub so ntswg: Nyhav (qhuav): ≥1 g
* Rau cov ntaub so ntswg me dua 5 mg, peb pom zoo kom xa flash khov (hauv kua nitrogen) cov ntaub so ntswg.
Cell suspension: Cell suav = 3 × 107
* Peb pom zoo kom nkoj khov cell lysate.Yog hais tias lub xov tooj ntawm tes suav me dua 5 × 105, flash khov hauv kua nitrogen yog pom zoo.
Cov ntshav kuaj:
PA × geneBloodRNATube;
6 mLTRIzol thiab 2 mL ntshav (TRIzol: Ntshav = 3: 1)
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 muab 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
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
Bioinformatics
1.LncRNA kev faib tawm
LncRNA kwv yees los ntawm plaub softwares saum toj no tau muab faib ua 4 pawg: lincRNA, anti-sense-LncRNA, intronic-LncRNA;kev-LncRNA.LncRNA kev faib tawm tau pom nyob rau hauv histogram hauv qab no.
LncRNA kev faib tawm
2.Cis-targeted genes ntawm DE-lncRNA enrichment tsom xam
ClusterProfiler tau ua haujlwm nyob rau hauv GO enrichment tsom xam ntawm cis-targeted noob ntawm txawv qhia lncRNA (DE-lncRNA), hais txog cov txheej txheem lom neeg, molecular zog thiab cellular Cheebtsam.GO enrichment tsom xam yog txheej txheem los txheeb xyuas DEG-qhia kom muaj txiaj ntsig GO cov ntsiab lus piv rau tag nrho cov genome.Cov ntsiab lus enriched tau nthuav tawm hauv histogram, daim duab npuas, thiab lwm yam raws li qhia hauv qab no.
Cis-targeted noob ntawm DE-lncRNA enrichment analysis -Bubble chart
3. Los ntawm kev sib piv qhov ntev, tus lej exon, ORF thiab kev nthuav qhia tus nqi ntawm mRNA thiab lncRNA, peb tuaj yeem nkag siab qhov sib txawv ntawm cov qauv, ntu thiab lwm yam ntawm lawv, thiab tseem xyuas seb qhov tshiab lncRNA kwv yees los ntawm peb ua raws li cov yam ntxwv dav dav.
BMK Cas
Deregulated lncRNA qhia profile hauv nas ntsws adenocarcinomas nrog KRAS-G12D kev hloov pauv thiab P53 knockout
Luam tawm:Phau ntawv Journal ntawm Cellular thiab Molecular Tshuaj,2019
Sequencing tswv yim
Illumina
Qauv sau
Lub NONMMUT015812-knockdown KP (shRNA-2) hlwb thiab kev tswj tsis zoo (sh-Scr) hlwb tau txais nyob rau hnub 6 ntawm tus kab mob tshwj xeeb.
Cov txiaj ntsig tseem ceeb
Txoj kev tshawb no tshawb xyuas qhov tsis zoo ntawm lncRNAs hauv nas ntsws adenocarcinoma nrog P53 knockout thiab KrasG12D kev hloov pauv.
1.6424 lncRNAs tau qhia txawv txawv (≥ 2-fold hloov, P <0.05).
2.Ntawm tag nrho 210 lncRNAs (FC≥8), 11 lncRNAs 'kev qhia tau tswj hwm los ntawm P53, 33 lncRNAs los ntawm KRAS thiab 13 lncRNAs los ntawm hypoxia hauv thawj KP hlwb, feem.
3.NONMMUT015812, uas tau zoo kawg li tswj hwm hauv nas ntsws adenocarcinoma thiab tswj tsis zoo los ntawm P53 rov qhia dua, tau tshawb pom los tshuaj xyuas nws txoj haujlwm ntawm tes.
4.Knockdown ntawm NONMMUT015812 los ntawm shRNAs txo qis proliferation thiab migration peev xwm ntawm KP hlwb.NONMMUT015812 yog qhov muaj peev xwm oncogene.
 KEGG txoj kev tsom xam ntawm qhov sib txawv qhia cov noob hauv NONMMUT015812-knockdown KP hlwb |  Gene Ontology tsom xam ntawm qhov sib txawv qhia cov noob hauv NONMMUT015812-knockdown KP hlwb |
Kev siv
Deregulated lncRNA qhia profile hauv nas ntsws adenocarcinomas nrog KRAS-G12D kev hloov pauv thiab P53 knockout [J].Phau ntawv Journal of Cellular thiab Molecular Medicine, 2019, 23(10).DOI: 10.1111/jcmm.14584
