Imikhiqizo

[Ikhophi] I-Eukaryotic mRNA Sequencing-NGS

[Ikhophi] Isithombe Esifakiwe se-Eukaryotic mRNA Sequencing-NGS

Ukulandelana kwe-mRNA, ubuchwepheshe obuguquguqukayo, kunika amandla ukuqoshwa okuphelele kwazo zonke izinguqulo ze-mRNA ngaphakathi kwamaseli ngaphansi kwezimo ezithile. Ngezinhlelo zayo ezibanzi, leli thuluzi eliphambili lembula amaphrofayili okubonakaliswa kwezakhi zofuzo ayinkimbinkimbi, izakhiwo zezakhi zofuzo, kanye nezinqubo zama-molecule ezihlotshaniswa nezinqubo ezahlukahlukene zebhayoloji. Njengoba yamukelwa kabanzi ocwaningweni oluyisisekelo, ekuxilongweni kwezokwelapha, kanye nasekuthuthukisweni kwemithi, ukulandelana kwe-mRNA kunikeza ukuqonda ngobunzima be-dynamics yamaseli kanye nokulawulwa kwezakhi zofuzo, okuvusa ilukuluku mayelana namandla ayo emikhakheni eyahlukene.

Ipulatifomu: Illumina NovaSeq X; DNBSEQ-T7

Xhumana nathi

Imininingwane Yesevisi

I-Bioinformatics

Imiphumela Yedemo

Izincwadi Ezivelele

Izici

● Ukuthwebula i-poly mRNA ngaphambi kokulungiselela umtapo wolwazi

● Ukulungiselela umtapo wezincwadi we-mRNA oqondiswayo ongakukhetha ukuze kuvunyelwe ukuthola idatha yokulandelana kwemigqa ethile

● Ukuhlaziywa kwe-Bioinformatic kokubonakaliswa kwezakhi zofuzo kanye nesakhiwo sombhalo oqoshiwe

Izinzuzo

●Ubuchwepheshe Obubanzi: Ithimba lethu licubungule amasampula angaphezu kuka-600,000 kwa-BMKgene, ahlanganisa izinhlobo ezahlukene zamasampula ezifana namasiko amaseli, izicubu, kanye noketshezi lomzimba. Siletha ulwazi oluningi kuyo yonke iphrojekthi futhi siqede ngempumelelo amaphrojekthi e-mRNA-Seq angaphezu kuka-100,000 emikhakheni eyahlukene yocwaningo.

●Ukulawulwa Kwekhwalithi Okuqinile: Sisebenzisa amaphuzu okulawula ayisisekelo kuzo zonke izigaba, kusukela ekulungiseleleni isampula kanye nomtapo wolwazi kuya ekulandeleni nasekubhaleni ulwazi nge-bioinformatics. Lokhu kuqapha ngokucophelela kuqinisekisa ukulethwa kwemiphumela esezingeni eliphezulu njalo, okuqinisekisa ukuthi iphrojekthi yakho isezandleni ezithembekile.

●Isichasiselo Esiphelele: Sisebenzisa izizindalwazi eziningi ukuze sichaze ngokusebenza i-Differentially Expressed Genes (DEGs) futhi senze ukuhlaziywa kokunotha okuhambisanayo. Le ndlela ebanzi inikeza ukuqonda ngezinqubo zamaseli kanye nama-molecule ezisekela impendulo ye-transcriptome, okuqinisekisa ukuthi unolwazi oluphelele ngemiphumela yephrojekthi yakho.

●Usekelo Lwangemva Kokuthengisa: Ukuzibophezela kwethu kudlulela ngale kokuqedwa kwephrojekthi ngesikhathi sesevisi yezinyanga ezintathu ngemuva kokuthengisa. Phakathi nalesi sikhathi, sinikeza ukulandelwa kwephrojekthi, usizo lokuxazulula izinkinga, kanye nezikhathi ze-Q&A ukuze kuxazululwe noma yimiphi imibuzo ehlobene nemiphumela.

Izidingo Zesampula Nokulethwa

Umtapo Wolwazi

Isu lokulandelana kwezigaba

Kunconywa idatha

Ikhwalithi yokulawula

I-Poly A ecebile

I-Illumina PE150

I-DNBSEQ-T7

6 -10 Gb

Q30≥85%

Izidingo Zesampula:

Ama-Nucleotide:

Izidingo Zesampula Ezincane:

I-Conc.(ng/μl)

Inani (μg)

Ubumsulwa

Ubuqotho

Umtapo Wolwazi Ojwayelekile

≥ 10

≥ 0.2

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Ukungcoliswa kwephrotheni noma i-DNA okulinganiselwe noma okungekho nhlobo kukhonjisiwe kujeli.

Kwezitshalo: I-RIN≥4.0;

Ezilwaneni: I-RIN≥4.5;

5.0≥28S/18S≥1.0;

ukuphakama okulinganiselwe noma okungekho kwesisekelo

Umtapo Wolwazi Oqondisayo

≥ 10

≥ 0.2

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Ukungcoliswa kwephrotheni noma i-DNA okulinganiselwe noma okungekho nhlobo kukhonjisiwe kujeli.

Kwezitshalo: I-RIN≥4.0;

Ezilwaneni: I-RIN≥4.5;

5.0≥28S/18S≥1.0;

ukuphakama okulinganiselwe noma okungekho kwesisekelo

Izitshalo

Isilwane

Ama-arthropod

Igazi eliphelele

Amaseli

Impande, Isiqu noma Ipetali: 450 mg

Iqabunga noma Imbewu: 300 mg

Izithelo: 1.2 g

Inhliziyo noma Amathumbu: 300 mg

I-Viscera noma Ubuchopho: 240 mg

Imisipha: 450 mg

Amathambo, Izinwele noma Isikhumba: 1g

Izinambuzane: 6g

I-Crustacea: 300 mg

Ishubhu eli-1

Amaseli angu-106

Ukulethwa Kwesampula Okunconyiwe

Isitsha: Ishubhu le-centrifuge elingu-2 ml (Akukhuthazwa ukusebenzisa i-tin foil)

Ukulebula kwesampula: Iqembu + ukukopisha isib. A1, A2, A3; B1, B2, B3... ...

Ukuthunyelwa:

1. Iqhwa elomile: Amasampula kudingeka apakishwe ezikhwameni bese egqitshwa eqhweni elomile.

2. Amashubhu etafula lama-RNA: Amasampula e-RNA angomiswa eshubhu lokuzinzisa i-RNA (isb. i-RNAstable®) bese ethunyelwa ekushiseni kwegumbi.

Izidingo Zesampula Nokulethwa

Buka Okuningi

Uma unemibuzo mayelana nemikhiqizo yethu noma uhlu lwamanani, sicela usishiyele i-imeyili yakho.
Sizoxhumana nathi zingakapheli amahora angama-24.

Uphenyo Manje

Okwedlule: I-Exosomal mRNA/LncRNA/CirrcRNA Sequencing-Illumina

Olandelayo: I-Metabolomics

I-Bioinformatics

I-Eukaryotic umsebenzi wokuhlaziya ukulandelana kwe-mRNA

I-Bioinformatics

ØUkulawulwa kwekhwalithi yedatha eluhlaza

ØUkulungiswa kwe-genome okubhekiselwe kuyo

ØUkuhlaziywa kwesakhiwo sombhalo oqoshiwe

ØUkulinganiswa kokubonakaliswa

ØUkuhlaziywa kokubonakaliswa kokwehluka

ØIsichasiselo somsebenzi kanye nokucebisa

Ukuqondaniswa Kwe-Genome Yereferensi

Ukugcwala kwedatha

Ukuhambisana kwesampula kanye nokuhlolwa kokuphindaphindwa kwezinto eziphilayo

Izakhi Zofuzo Ezivezwe Ngokuhlukile (ama-DEG)

Isichasiselo Esisebenzayo sama-DEG

Ukuthuthuka Okusebenzayo kwama-DEG

Hlola intuthuko eyenziwe yizinsizakalo zokulandelana kwe-BMKGene ze-eukaryotic NGS mRNA ngokusebenzisa iqoqo lezincwadi ezikhethiwe.

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UWen, X. et al. (2022) 'I-genome ye-Chrysanthemum lavandulifolium kanye nendlela esebenza ngayo ama-molecule engaphansi kwezinhlobo ezahlukene ze-capitulum', i-Horticulture Research, 9. doi: 10.1093/HR/UHAB022.

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