Ubude obupheleleyo be-mRNA Ukulandelelana-Nanopore
Iinkonzo eziluncedo
● I-bias yolandelelwano oluphantsi
● Ukutyhila iimolekyuli ze-cDNA ezipheleleyo
● Idatha encinci efunekayo ukugubungela inani elifanayo lemibhalo ebhaliweyo
● Ukuchongwa kwee-isoform ezininzi kwijini ngalinye
● Ubungakanani benkcazo kwinqanaba le-isoform
IiNgcaciso zeNkonzo
| Ithala leencwadi | Iqonga | Imveliso yedatha ecetyiswayo (Gb) | Ulawulo lwemeko |
| cDNA-PCR(Poly-A enriched) | Nanopore PromethION P48 | 6 Gb/isampulu (Kuxhomekeke kwiintlobo) | Umlinganiselo wobude obupheleleyo＞70% Amanqaku omgangatho ophakathi: Q10
|
Uhlalutyo lwe-Bioinformatics
●Ukusetyenzwa kwedatha ekrwada
● Ukuchongwa okubhaliweyo
● Enye indlela yokudibanisa
● Ubungakanani benkcazo kwinqanaba le-gene kunye nenqanaba le-isoform
● Uhlalutyo lwentetho eyahlukileyo
● Inkcazo yomsebenzi kunye nokutyebisa (iiDEGs kunye neeDETs)
IiMfuno zeSampulu kunye nokuhanjiswa
IiMfuno zeSampulu:
IiNucleotides:
| Conc.(ng/μl) | Imali (μg) | Ubunyulu | Ingqibelelo |
| ≥ 100 | ≥ 0.6 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Unyino okanye akukho protein okanye ungcoliseko lweDNA olubonakaliswe kwijeli. | Kwizityalo: RIN≥7.0; Kwizilwanyana: RIN≥7.5; 5.0≥28S/18S≥1.0; umphakamo osisiseko okanye ongekhoyo |
Izicubu: Ubunzima (obomileyo): ≥1 g
*Kwithishu ezincinci kuno-5 mg, sicebisa ukuba uthumele ifleshi ekhenkcezisiweyo (kwinitrogen engamanzi) isampulu yethishu.
Ukumiswa kweeseli: Ubalo lweeseli = 3×106- 1×107
*Sicebisa ukuthumela i-lysate yeseli enomkhenkce.Kwimeko apho iseli ibala encinci kune-5 × 105, i-flash ekhenkcezisiweyo kulwelo lwenitrogen iyacetyiswa, ekhethwayo xa kutsalwa kancinci.
Iisampuli zegazi: Umthamo≥1 ml
Unikezelo lweSampuli eCetyisiweyo
Isikhongozeli: 2 ml centrifuge ityhubhu (Ifoyile yeTin ayikhuthazwa)
Isampuli yokuleyibhela: Iqela+liphindaphinda umz. A1, A2, A3;B1, B2, B3... ...
Ukuthunyelwa: 2, Umkhenkce owomileyo: Iisampuli kufuneka zipakishwe kwiingxowa kwaye zingcwatywe kwindawo eyomileyo.
- I-RNAstable tubes: Iisampulu ze-RNA zingomiswa kwi-RNA stabilization tube (umzekelo, i-RNAstable®) kwaye ithunyelwe kwiqondo lokushisa.
Ukuhamba komsebenzi weNkonzo
IiNucleotides:
Ukuhanjiswa kwesampuli
Ukwakhiwa kwethala leencwadi
Ukulandelelanisa
Uhlalutyo lwedatha
Iinkonzo emva kokuthengisa
Ukuhamba komsebenzi weNkonzo
Izicubu:
Uyilo lovavanyo
Ukuhanjiswa kwesampuli
Ukukhutshwa kwe-RNA
Ukwakhiwa kwethala leencwadi
Ukulandelelanisa
Uhlalutyo lwedatha
Iinkonzo emva kokuthengisa
1.Uhlalutyo lwentetho eyahlukileyo -isakhiwo seVolcano
Uhlalutyo lwentetho eyohlukeneyo lunokucutshungulwa kuwo omabini amanqanaba emfuza ukuchonga imfuza echazwe ngokwahlukileyo (DEGs) nakwinqanaba le-isoform ukuchonga ngokwahlukileyo.
imibhalo eshicilelweyo (DETs)
2.Imephu yobushushu ehlanganisana ngokwemigangatho
3.Ukuchongwa kwe-Alternative splicing kunye nokuhlelwa
Iindidi ezintlanu zeziganeko ezizezinye zokudibanisa zinokuqikelelwa yi-Astalavista.
4.Enye iPoly-adenylation (APA) ukuchongwa kweziganeko kunye neMotif ku-50 bp phezulu komsinga wepoly-A
Ityala le-BMK
Uchongo olulolunye lwe-splicing kunye ne-isoform-level quantification yi-nanopore yobude obugcweleyo bokulandelelana kwe-transcriptome
Ipapashiwe:Unxibelelwano ngeNdalo, ngo-2020
Iqhinga lokulandelelanisa:
Ukwahlulahlula: 1. CLL-SF3B1(WT);2. CLL-SF3B1(K700E ukuguqulwa);3. Iiseli ze-B eziqhelekileyo
Iqhinga lolandelelwano: MiniION 2D ithala leencwadi ngolandelelwano, PromethION 1D ulandelelwano lwethala leencwadi;idatha yokufunda emfutshane kwiisampuli ezifanayo
Iqonga lokulandelelana: I-Nanopore MinION;I-Nanopore PromethION;
Iziphumo eziphambili
1.Inqanaba le-Isoform-level ye-Alternative Splicing Identification
Ulandelelwano olufunda ixesha elide luxhobisa ukuchongwa kwe-SF3B1 eguqukayoK700E-altered splice sites at isoform-level.Ngama-35 ezinye ii-3′SS kunye ne-10 ezizezinye ezi-5′SS zifunyenwe zicandeke kakhulu ngokwahlukileyo phakathi kweSF3B1K700Ekunye neSF3B1WT.I-33 kwezingama-35 ezitshintshiweyo zisanda kufunyanwa ngolandelelwano lokufunda ixesha elide.
2.Inqanaba le-Isoform ye-Alternative Splicing quantification
Ukuchazwa kwe-intron retention(IR) isoforms kwi-SF3B1K700Ekunye neSF3B1WTZiye zabalwa ngokusekwe kulandelelwano lwe-nanopore, lutyhila ukulawulwa kwehlabathi jikelele kwe-IR isoforms kwi-SF3B1K700E.
Isalathiso
Tang AD , Soulette CM , Baren MJV , et al.Ubude obupheleleyo be-transcript characterization ye-SF3B1 utshintsho kwi-lymphocytic leukemia engapheliyo ibonisa ukuthotywa kwe-introns ezigciniweyo[J].Unxibelelwano lweNdalo.
