Imikhiqizo

I-Eukaryotic mRNA Sequencing-Illumina

Ukulandelana kwe-mRNA kunika amandla ukuphrofayela wawo wonke ama-mRNA alotshwe kusuka kumaseli ngaphansi kwezimo ezithile.Ubuchwephesha obunamandla bokuveza iphrofayili yesisho sofuzo, izakhiwo zofuzo nezindlela zamangqamuzana ezinqubo ezithile zebhayoloji.Kuze kube manje, ukulandelana kwe-mRNA sekusetshenziswe kabanzi ocwaningweni oluyisisekelo, ekuxilongweni komtholampilo, ekuthuthukisweni kwezidakamizwa, njll.

Ipulatifomu: I-Illumina NovaSeq Platform

Xhumana nathi

Imininingwane Yesevisi

I-Bioinformatics

Imiphumela yedemo

Izinzuzo

● Abanolwazi Kakhulu: Amasampula angaphezu kuka-200,000 acutshungulwe ku-BMK ehlanganisa izinhlobo zamasampula ahlukahlukene, okuhlanganisa isiko lamaseli, izicubu, uketshezi lomzimba, njll. kanye namaphrojekthi angaphezu kuka-7,000 mRNA-Seq avaliwe ahlanganisa izindawo ezihlukahlukene zocwaningo.

● Isistimu yokulawula ikhwalithi eqinile: Amaphuzu okulawula ikhwalithi abalulekile ngazo zonke izinyathelo ezihlanganisa ukulungiswa kwesampula, ukulungiswa komtapo wolwazi, ukulandelana kanye ne-bioinformatics angaphansi kokuqapha ukuze kulethwe imiphumela yekhwalithi ephezulu.

● Imininingo egciniwe eminingi etholakalayo yesichasiselo somsebenzi kanye nezifundo zokucebisa ukufeza izinjongo zocwaningo ezihlukahlukene.

● Amasevisi angemuva kokuthengisa: Amasevisi angemuva kokuthengisa asebenza izinyanga ezi-3 ngemva kokuqedwa kwephrojekthi, okuhlanganisa ukulandelela amaphrojekthi, ukuxazulula izinkinga, imiphumela ye-Q&A, njll.

Izidingo Zesampula Nokulethwa

Umtapowolwazi	Isu lokulandelanisa	Idatha iyanconywa	Ikhwalithi yokulawula
I-Poly A inothisiwe	I-Ilumina PE150	6 gb	Q30≥85%

Izimfuneko zesampula:

Ama-Nucleotides:

Conc.(ng/μl)

Inani (μg)

Ubumsulwa

Ubuqotho

≥ 20

≥ 0.5

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Amaprotheni anomkhawulo noma awekho nhlobo noma ukungcola kwe-DNA okuboniswa kujeli.

Okwezitshalo: RIN≥6.5;

Okwezilwane: RIN≥7.0;

5.0≥28S/18S≥1.0;

ukuphakama okulinganiselwe noma okungenakho

Izicubu: Isisindo(somile):≥1 g
*Ngezicubu ezincane kuno-5 mg, sincoma ukuthi uthumele isampula yezicubu eziqandisiwe (ku-nitrogen ewuketshezi).

Ukumiswa kweseli:Inani lamaseli = 3×10⁶- 1 × 10⁷
*Sincoma ukuthi uthumele i-frozen cell lysate.Esimeni lapho lelo seli liba lincane kuno-5×105.

Amasampula egazi:Umthamo≥1 ml

I-Microorganism:Isisindo ≥ 1 g

Ukulethwa Kwesampula Okunconyiwe

Isitsha: 2 ml centrifuge tube (Tin foil ayinconywa)

Ukulebula okuyisampula: Ukuphindaphinda kweqembu+isb A1, A2, A3;B1, B2, B3....

Ukuthunyelwa:

Iqhwa elomile: Amasampula adinga ukupakishwa ezikhwameni futhi angcwatshwe eqhweni elomile.
Amashubhu e-RNAstable: Amasampula e-RNA angomiswa ngeshubhu lokuzinzisa le-RNA(isb. i-RNAstable®) futhi athunyelwe ngegumbi lokushisa.