Prokaryotic RNA Sequencing
Nta
● RNA cov qauv ua piv txwv koom nrog rRNA depletion ua raws li kev taw qhia RNA tsev qiv ntawv npaj.
● Bioinformatic tsom xam raws li kev ua kom haum rau ib qho kev siv genome
● Kev soj ntsuam suav nrog cov noob qhia thiab DEGs tab sis kuj muaj cov qauv sau ntawv thiab kev tshuaj xyuas sRNA
Kev Pabcuam Zoo
●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.
●Strand-specific Sequencing Data: vim yog lub tsev qiv ntawv RNA npaj tau coj mus, ua kom muaj kev txheeb xyuas cov ntaub ntawv tiv thaiv kev nkag siab.
●Ua kom tiav Kev Ntsuam Xyuas Ua tiav rau Prokaryotic Transcriptomes: cov raj xa dej bioinformatic suav nrog tsis yog tsuas yog kev tshuaj xyuas ntawm cov noob qhia tab sis kuj tseem tshuaj xyuas cov qauv sau ntawv, suav nrog kev txheeb xyuas cov neeg ua haujlwm, UTRs thiab cov neeg txhawb nqa. Nws kuj suav nrog kev txheeb xyuas ntawm sRNAs, uas yog cov lus piav qhia thiab kev kwv yees ntawm cov qauv thib ob thiab cov hom phiaj.
●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 |
| rRNA depleted directional tsev qiv ntawv | Illumina PE 150 | 1-2 Gb | Q30≥85% |
Cov Qauv Xav Tau:
| Conc.(ng/μl) | Tus nqi (μg) | Purity | Kev ncaj ncees |
| ≥ 50 | ≥ 1 | OD 260/280 = 1.8-2.0 OD 260/230 = 1.0-2.5 Txwv los yog tsis muaj protein ntau lossis DNA kis tau pom ntawm gel. | RIN ≥6.5 |
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
Qauv xa tuaj
Kev tsim kho tsev qiv ntawv
Sequencing
Cov ntaub ntawv tsom xam
Kev pab cuam tom qab muag
Bioinformatic Analysis Workflow
Xws li Kev Tshawb Fawb Hauv qab no:
● Raw cov ntaub ntawv zoo tswj
● Alignment rau lub genome siv
● Kev Ntsuam Xyuas Lub Tsev Qiv Ntawv Zoo: RNA fragmentation randomness, ntxig qhov loj thiab sequencing saturation
● Muaj nuj nqi annotation ntawm kwv yees coding noob
● Kev Ntsuam Xyuas Kev Tshaj Tawm: Kev sib raug zoo thiab Kev Ntsuas Tus Thawj Saib Xyuas Haujlwm (PCA)
● Differential Gene Expression (DEGs)
● Cov lus piav qhia ua haujlwm thiab ua kom muaj txiaj ntsig ntawm DEGs
● sRNA Kev Ntsuam Xyuas: kev twv ua ntej, annotation, lub hom phiaj, thiab kev kwv yees tus qauv theem nrab
● Kev Ntsuam Xyuas Cov Qauv Tshaj Lij Tshaj Lij: Cov neeg ua haujlwm, pib thiab xaus txoj haujlwm, cheeb tsam Untranslated (UTS), tus txhawb nqa, thiab SNP/InDel tsom xam
Sequencing saturation
Functional annotation ntawm coding noob
Kev sib raug zoo ntawm cov qauv
Differential Expressed Genes (DEGs) tsom xam
Functional enrichment tsom xam
sRNA annotation
Tshawb nrhiav cov kev nce qib uas tau pab los ntawm BMKGene's Nanopore tag nrho-ntev mRNA sequencing cov kev pabcuam hauv cov ntawv tshaj tawm no.
Guan, CP et al. (2018) 'Ntiaj teb Transscriptome Hloov ntawm Biofilm-Forming Staphylococcus epidermidis Teb rau Tag Nrho Alkaloids ntawm Sophorea alopecuroides',Polish Journal of Microbiology, 67(2), p. 223. doi: 10.21307/PJM-2018-024.
