Kev Tshawb Fawb Txog MRNA Tag Nrho-Nanopore
Cov yam ntxwv
● Kev ntes cov poly-A mRNA ua raws li kev tsim cDNA thiab kev npaj tsev qiv ntawv
● Kev txheeb xyuas cov ntawv sau tag nrho
● Kev tshuaj xyuas bioinformatic raws li kev sib phim rau cov genome siv
● Kev tshuaj xyuas bioinformatic tsis yog tsuas yog suav nrog kev qhia tawm ntawm theem gene thiab isoform xwb tab sis kuj tseem muaj kev tshuaj xyuas ntawm lncRNA, gene fusions, poly-adenylation thiab gene structure
Cov Kev Pabcuam Zoo
●Kev ntsuas ntawm kev qhia tawm ntawm theem isoform: ua kom muaj kev tshuaj xyuas kev qhia tawm kom meej thiab raug, qhia txog kev hloov pauv uas yuav raug zais thaum tshuaj xyuas tag nrho cov noob caj noob ces
●Txo Cov Kev Xav Tau Cov Ntaub Ntawv:Piv rau Next-Generation Sequencing (NGS), Nanopore sequencing qhia txog cov ntaub ntawv qis dua, uas tso cai rau cov qib sib npaug ntawm kev ntsuas kev qhia txog gene nrog cov ntaub ntawv me dua.
●Kev ntsuas qhov tseeb ntawm kev qhia tawm ntau dua: ob qho tib si ntawm theem gene thiab isoform
●Kev txheeb xyuas cov ntaub ntawv sau ntxiv: lwm txoj kev polyadenylation, fusion genes thiab lcnRNA thiab lawv cov target genes
●Kev Paub Ntau YamPeb pab neeg coj ntau yam kev paub dhau los rau txhua qhov project, tau ua tiav ntau dua 850 Nanopore cov haujlwm transcriptome puv ntoob thiab ua tiav ntau dua 8,000 cov qauv.
●Kev Txhawb Nqa Tom Qab Muag Khoom: peb txoj kev cog lus txuas ntxiv mus dhau qhov ua tiav qhov project nrog rau 3 lub hlis tom qab muag kev pabcuam. Thaum lub sijhawm no, peb muab kev saib xyuas qhov project, kev pab daws teeb meem, thiab cov lus nug thiab lus teb los teb cov lus nug ntsig txog cov txiaj ntsig.
Cov Qauv Yuav Tsum Tau Ua thiab Kev Xa Khoom
| Tsev qiv ntawv | Txoj kev npaj ua sequencing | Cov ntaub ntawv pom zoo | Kev Tswj Xyuas Zoo |
| Poly A nplua nuj | Nanopore PromethION 48 | 6/12 Gb | Qhov qhab nia zoo nruab nrab: Q10 |
Cov Qauv Yuav Tsum Tau Ua:
Cov nucleotides:
| Conc.(ng/μl) | Tus nqi (μg) | Kev dawb huv | Kev ncaj ncees |
| ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Muaj tsawg los yog tsis muaj protein lossis DNA uas pom ntawm gel. | Rau cov nroj tsuag: RIN≥7.0; Rau cov tsiaj: RIN≥7.5; 5.0≥28S / 18S≥1.0; txwv los yog tsis muaj qhov siab pib |
● Cov Nroj Tsuag:
Cag, Qia los yog Paj: 450 mg
Nplooj los yog noob: 300 mg
Txiv hmab txiv ntoo: 1.2 g
● Tsiaj:
Lub plawv los yog hnyuv: 300 mg
Cov hnyuv lossis lub hlwb: 240 mg
Cov leeg: 450 mg
Pob txha, Plaub hau los yog Tawv Nqaij: 1g
● Cov kab mob Arthropod:
Kab: 6g
Cov ntses me me: 300 mg
● Cov ntshav tag nrho: 1 lub raj
● Cov Cell: 106 cov hlwb
Kev Xa Qauv Pom Zoo
Lub thawv: 2 ml centrifuge tube (Tsis pom zoo kom siv ntawv ci)
Piv txwv kev sau npe: Pawg + theej piv txwv li A1, A2, A3; B1, B2, B3.
Kev Xa Khoom:
1. Dej khov qhuav: Cov qauv yuav tsum tau ntim rau hauv hnab thiab faus rau hauv dej khov qhuav.
2. Cov raj RNAstable: Cov qauv RNA tuaj yeem ziab hauv cov raj RNA stabilization (piv txwv li RNAstable®) thiab xa tuaj rau hauv chav tsev kub.
Kev Ua Haujlwm Pabcuam
Cov nucleotides:
Kev xa khoom qauv
Kev tsim kho tsev qiv ntawv
Kev txheeb xyuas kab ke
Kev tshuaj xyuas cov ntaub ntawv
Cov kev pab cuam tom qab muag
Kev Ua Haujlwm Pabcuam
Cov ntaub so ntswg:
Tsim qauv kev sim
Kev xa khoom qauv
Kev rho tawm RNA
Kev tsim kho tsev qiv ntawv
Kev txheeb xyuas kab ke
Kev tshuaj xyuas cov ntaub ntawv
Cov kev pab cuam tom qab muag
● Kev ua cov ntaub ntawv raw
● Kev txheeb xyuas cov ntawv sau tseg
● Kev sib txuas lwm txoj kev
● Kev ntsuas kev qhia tawm hauv theem gene thiab theem isoform
● Kev tshuaj xyuas qhov sib txawv ntawm kev qhia tawm
● Kev piav qhia txog kev ua haujlwm thiab kev txhim kho (DEGs thiab DETs)
Kev tshuaj xyuas lwm txoj hauv kev sib txuas
Kev Tshawb Fawb Txog Polyadenylation Lwm Txoj (APA)
kev kwv yees lncRNA
Kev piav qhia ntawm cov noob caj noob ces tshiab
Kev sib sau ua ke ntawm DETs
Cov Protein-Protein Networks hauv DEGs
Tshawb nrhiav cov kev nce qib uas tau pab txhawb los ntawm BMKGene's Nanopore full-length mRNA sequencing cov kev pabcuam los ntawm kev sau cov ntawv tshaj tawm.
Gong, B. et al. (2023) 'Epigenetic thiab transcriptional activation ntawm secretory kinase FAM20C ua ib qho oncogene hauv glioma', Journal of Genetics and Genomics, 50(6), pp. 422–433. doi: 10.1016/J.JGG.2023.01.008.
He, Z. et al. (2023) 'Kev txheeb xyuas cov kab mob lymphocytes tag nrho rau IFN-γ qhia txog kev tiv thaiv kab mob Th1-skewed hauv flounder (Paralichthys olivaceus)', Fish & Shellfish Immunology, 134, p. 108636. doi: 10.1016/J.FSI.2023.108636.
Ma, Y. et al. (2023) 'Kev tshuaj xyuas sib piv ntawm PacBio thiab ONT RNA sequencing txoj kev rau Nemopilema Nomurai venom kev txheeb xyuas', Genomics, 115(6), p. 110709. doi: 10.1016/J.YGENO.2023.110709.
Yu, D. et al. (2023) 'Kev tshuaj xyuas Nano-seq qhia txog kev sib txawv ntawm kev ua haujlwm ntawm exosomes thiab microvesicles uas tau los ntawm hUMSC', Stem Cell Research and Therapy, 14(1), pp. 1–13. doi: 10.1186/S13287-023-03491-5/TABLES/6.
