Tsis siv neeg raws li mRNA Sequencing-Illumina
Nta
● Kev ywj pheej ntawm ib qho kev siv genome,
● Cov ntaub ntawv tuaj yeem siv los txheeb xyuas cov qauv thiab kev qhia ntawm cov ntawv sau tseg
● Txheeb xyuas qhov chaw clipping sib txawv
Kev Pabcuam Zoo
● BMKCloud-raws li cov txiaj ntsig xa tawm: Cov txiaj ntsig tau xa tawm raws li cov ntaub ntawv thiab kev sib tham sib qhia ntawm BMKCloud platform, uas tso cai rau cov neeg siv kev nyeem ntawv ntawm cov kev txheeb xyuas nyuaj thiab kev kho cov ntaub ntawv mining los ntawm cov qauv bioinformatics tsom xam.
● Cov kev pab cuam tom qab muag: Cov kev pab cuam tom qab muag siv tau rau 3 lub hlis tom qab ua tiav qhov project, suav nrog cov phiaj xwm rov qab, kev daws teeb meem, cov txiaj ntsig Q&A, thiab lwm yam.
Sample Requirements thiab Delivery
Cov Qauv Xav Tau:
Nucleotides:
| Conc. (ng / μl) | Tus nqi (μg) | Purity | Kev ncaj ncees |
| ≥ 20 | ≥ 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 |
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 raj (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.mRNA(denovo) Principple of Assembly
Los ntawm Trinity, nyeem tau muab faib ua tej daim me me, hu ua K-mer.Cov K-mers no yog siv los ua cov noob kom txuas mus rau hauv contigs thiab tom qab ntawd cov khoom siv raws li contig overlappings.Thaum kawg, De Bruijn tau thov ntawm no kom paub txog cov ntawv sau tseg hauv cov khoom.
mRNA (De novo) Txheej txheem cej luam ntawm Trinity
2.mRNA (De novo) Distribution of Gene Expression Level
RNA-Seq muaj peev xwm ua tiav qhov kev kwv yees zoo heev ntawm cov noob qhia.Nquag, qhov ntsuas tau ntau ntawm cov ntaub ntawv sau tseg FPKM yog nyob ntawm 10^-2 txog 10^6.
mRNA (De novo) Kev faib tawm ntawm FPKM ceev hauv txhua tus qauv
3.mRNA (De novo) GO Enrichment Analysis of DEGs
GO (Gene Ontology) database yog cov txheej txheem kev piav qhia lom neeg uas muaj cov ntsiab lus ntawm cov noob thiab cov khoom lag luam.Nws muaj ntau theem, qhov twg qib qis dua, qhov tshwj xeeb tshaj yog cov haujlwm.
mRNA (De novo) GO kev faib tawm ntawm DEGs ntawm qib thib ob
BMK Cas
Transcriptome Analysis ntawm Sucrose Metabolism thaum Lub Teeb o thiab Kev Loj Hlob hauv Dos (Allium cepa L.)
Luam tawm: frontiers nyob rau hauv cog scienceXyoo 2016
Sequencing tswv yim
Illumina HiSeq 2500
Qauv sau
Utah Yellow Sweet Spain cultivar "Y1351" tau siv hauv txoj kev tshawb no.Tus naj npawb ntawm cov qauv sau tau
15 hnub tom qab o (DAS) ntawm qhov muag teev (2-cm txoj kab uas hla thiab 3-4 g hnyav), 30th DAS (5-cm txoj kab uas hla thiab 100-110 g hnyav), thiab ∼3 ntawm 40th DAS (7-cm txoj kab uas hla thiab 260-300 g).
Cov txiaj ntsig tseem ceeb
1. nyob rau hauv daim duab Venn, tag nrho ntawm 146 DEGs tau kuaj pom thoob plaws peb khub ntawm kev loj hlob theem
2. "Carbohydrate thauj thiab metabolism" tsuas yog 585 unigenes (piv txwv li, 7% ntawm cov lus piav qhia COG).
3.Unigenes ua tiav cov lus piav qhia rau GO database tau muab faib ua peb pawg thawj coj rau peb theem sib txawv ntawm kev txhim kho noob.Feem ntau sawv cev hauv "txoj kev lom neeg" pawg thawj coj yog "cov txheej txheem metabolic", ua raws li "cov txheej txheem ntawm tes".Hauv qeb tseem ceeb ntawm "kev ua haujlwm molecular" ob pawg feem ntau sawv cev yog "kev khi" thiab "kev ua haujlwm catalytic".
 Histogram ntawm pawg ntawm orthologous pawg (COG) kev faib tawm |  Histogram ntawm noob ontology (GO) kev faib tawm rau unigenes muab los ntawm qhov muag teev hauv peb theem kev loj hlob |
 Venn daim duab qhia cov noob sib txawv qhia nyob rau hauv ob theem ntawm kev loj hlob dos noob |
Kev siv
Zhang C, Zhang H, Zhan Z, et al.Transcriptome Analysis ntawm Sucrose Metabolism thaum Lub Teeb o thiab Kev Loj Hlob hauv Dos (Allium cepa L.)[J].Frontiers in Plant Science, 2016, 7:1425-.DOI: 10.3389/fpls.2016.01425
