Full-Length mRNA Sequencing-Nanopore
Kev Pabcuam Zoo
● Tsawg ib ntus kev tsis ncaj ncees
● nthuav tawm tag nrho-ntev cDNA molecules
● Cov ntaub ntawv tsawg dua yuav tsum tau them rau tib tus lej ntawm cov ntawv sau tseg
● Kev txheeb xyuas ntau yam isoforms ib lub noob
● Kev nthuav qhia kom muaj nuj nqis hauv qib isoform
Service Specifications
| Lub tsev qiv ntawv | Platform | Cov ntaub ntawv pom zoo yield (Gb) | Kev Tswj Xyuas Zoo |
| cDNA-PCR (Poly-A enriched) | Nanopor PromethION P48 | 6 Gb / qauv (nyob ntawm hom) | Tag nrho-length ratio> 70% Qhov qhab nia nruab nrab: Q10
|
Bioinformatics tsom xam
●Cov ntaub ntawv nyoos
● 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 nqa (DEGs thiab DETs)
Sample Requirements thiab Delivery
Cov Qauv Xav Tau:
Nucleotides:
| Conc. (ng / μl) | Tus nqi (μg) | Purity | Kev ncaj ncees |
| ≥100 | ≥ 0.6 | 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≥7.0; Rau tsiaj: RIN≥7.5; 5.0 ≥28S / 18S≥1.0; txwv los yog tsis muaj qhov nce siab |
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 × 106- 1 × 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, uas yog qhov zoo dua rau kev rho tawm micro.
Cov ntshav kuaj: Volume ≥ 1 ml
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: 2 、 Cov dej khov qhuav: Cov qauv yuav tsum tau ntim rau hauv hnab thiab faus rau hauv cov dej khov qhuav.
- RNAstable hlab: 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
1.Differential qhia kev tsom xam - Volcano zajlus
Kev txheeb xyuas qhov sib txawv tuaj yeem ua tiav hauv ob theem noob los txheeb xyuas qhov sib txawv ntawm cov noob (DEGs) thiab hauv qib isoform txhawm rau txheeb xyuas qhov sib txawv.
Cov ntawv tshaj tawm (DETs)
2.Hierarchical clustering heatmap
3.Alternative splicing kev txheeb xyuas thiab kev faib tawm
Tsib hom kev sib txuas lwm qhov xwm txheej tuaj yeem kwv yees los ntawm Astalavista.
4.Alternative poly-adenylation (APA) kev txheeb xyuas thiab Motif ntawm 50 bp upstream ntawm poly-A
BMK Cas
Alternative splicing identification thiab isoform-level quantification by nanopore full-length transcriptome sequencing
Luam tawm:Nature Communications, 2020
Sequence Strategy:
Pawg: 1. CLL-SF3B1(WT);2. CLL-SF3B1(K700E mutation);3. Ib txwm B-hlwb
Sequencing lub tswv yim: MinION 2D tsev qiv ntawv sequencing, PromethION 1D tsev qiv ntawv sequencing;luv-nyeem cov ntaub ntawv los ntawm tib cov qauv
Sequencing platform: Nanopore MinION;Nanopor PromethION;
Cov txiaj ntsig tseem ceeb
1.Isoform-level Alternative Splicing Identification
Kev nyeem ntawv ntev ua ntu zus txhawb kev txheeb xyuas cov mutant SF3B1K700 E- hloov chaw splice ntawm isoform-theem.35 lwm 3′SSs thiab 10 lwm 5′SSs tau pom tias muaj qhov sib txawv ntawm qhov sib txawv ntawm SF3B1K700 Ethiab SF3B1WT.33 ntawm 35 qhov kev hloov pauv tau raug tshawb pom tshiab los ntawm cov ntawv nyeem ntev.
2.Isoform-level Alternative Splicing quantification
Kev nthuav qhia ntawm intron retention (IR) isoforms hauv SF3B1K700 Ethiab SF3B1WTtau txheeb xyuas raws li nanopore sequences, nthuav tawm lub ntiaj teb down-txoj cai ntawm IR isoforms hauv SF3B1K700 E.
Kev siv
Tang AD, Soulette CM, Baren MJV, et al.Tag nrho-ntev transcript characterization ntawm SF3B1 kev hloov pauv hauv cov kab mob lymphocytic leukemia ntev qhia txog kev txo qis ntawm cov khoom khaws cia [J].Nature Communications.
