CircRNA sequencing-Illumina
Nta
● rRNA depletion ua raws li kev npaj cov tsev qiv ntawv qhia, ua kom cov ntaub ntawv sib txuas uas yog cov kab tshwj xeeb.
● Bioinformatic workflow enables circRNA kwv yees thiab qhia kom muaj nuj nqis
Kev Pabcuam Zoo
●Cov tsev qiv ntawv RNA ntau dua:peb siv rRNA depletion es tsis txhob linear RNA depletion nyob rau hauv peb pre-library npaj, xyuas kom meej tias cov sequencing cov ntaub ntawv muaj xws li tsis tsuas circRNA tab sis kuj mRNA thiab lncRNA, ua kom muaj kev sib koom tsom xam ntawm cov datasets.
●Kev txheeb xyuas ntawm kev sib tw endogenous RNA (ceRNA) tes hauj lwm: muab kev nkag siab tob rau hauv cellular tswj mechanisms
●Kev txawj ntse: nrog cov ntaub ntawv teev tseg ntawm kev ua tiav ntau dua 20,000 tus qauv ntawm BMK, nthuav dav ntau hom qauv thiab cov haujlwm lncRNA, peb pab pawg coj ntau yam kev paub rau txhua qhov haujlwm.
●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.
● 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 kev muag khoom.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 | Platform | Cov ntaub ntawv pom zoo | Cov ntaub ntawv QC |
| Poly A enriched | Illumina PE 150 | 16-20 Gb | Q30≥85% |
Cov Qauv Xav Tau:
Nucleotides:
| 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 |
● 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 lossis txoj hnyuv: 450 mg
Viscera lossis hlwb: 240 mg
Cov leeg: 600 mg
Pob txha, plaub hau lossis tawv nqaij: 1.5g
● Arthropods:
piv: 9g
Crustacea: 450 mg
● Cov ntshav tag nrho:2 cev
● Cells: 106 hlwb
● Ntshav thiab Plasmaib: 6ml
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
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
CircRNA twv ua ntej: chromosomal faib
Sib txawv qhia circRNAs - Volcano zajlus
Sib txawv qhia circRNAs - hierarchical pawg
Kev ua kom muaj txiaj ntsig zoo ntawm circRNA tus tswv tsev cov noob
Tshawb nrhiav cov kev tshawb fawb nce qib los ntawm BMKGene 'circRNA sequencing cov kev pabcuam los ntawm kev sau cov ntawv tshaj tawm.
Wang, X. et al.(2021) 'CPSF4 tswj kev tsim circRNA thiab microRNA mediated gene silencing hauv hepatocellular carcinoma', Oncogene 2021 40:25, 40(25), pp. 4338–4351.doi: 10.1038/s41388-021-01867-6.
Xia, K. et al.(2023) 'X oo-responsive transcriptome qhia lub luag haujlwm ntawm lub voj voog RNA133 hauv kev tiv thaiv kab mob los ntawm kev tswj hwm kev qhia ntawm OsARAB hauv mov', Phytopathology Research, 5(1), pp. 1–14.doi: 10.1186/S42483-023-00188-8/FIGURES/6.
Y, H. et al.(2023) 'CPSF3 modulates qhov sib npaug ntawm cov voj voog thiab cov ntawv sau tawm hauv cov kab mob hepatocellular'.doi: 10.21203/RS.3.RS-2418311/V1.
Zhang, Y. Y. et al.(2023) 'Comprehensive evaluation of circRNAs in cirrhotic cardiomyopathy ua ntej thiab tom qab hloov daim siab', International Immunopharmacology, 114, p.109495. doi: 10.1016/J.INTIMP.2022.109495.
