Iimveliso

Eukaryotic mRNA Sequencing-Illumina

Eukaryotic mRNA Ulandelelwano-Illumina Umfanekiso obonakalayo

Ulandelelwano lwe-mRNA lwenza ukuba iprofayili yazo zonke ii-mRNA zikhutshelwe kwiiseli phantsi kweemeko ezithile.Bubuchwephesha obunamandla bokutyhila inkangeleko yemfuza yemfuza, izakhiwo zemfuza kunye neendlela zemolekyuli zeenkqubo ezithile zebhayoloji.Ukuza kuthi ga ngoku, ulandelelwano lwe-mRNA lusetyenziswe ngokubanzi kuphando olusisiseko, uxilongo lwezonyango, uphuhliso lwamachiza, njl.

Iqonga: I-Illumina NovaSeq Platform

Qhagamshelana nathi

Iinkcukacha zeNkonzo

Bioinformatics

Iziphumo zeDemo

Iingenelo

● Amava aphezulu: Ngaphezu kwe-200,000 yeesampulu ezisetyenzisiweyo kwi-BMK egubungela iindidi ezahlukeneyo zeesampulu, kubandakanywa inkcubeko yeeseli, izicubu, ulwelo lomzimba, njl.njl. kunye neeprojekthi ezingaphezu kwe-7,000 mRNA-Seq ezivaliweyo ezibandakanya iindawo ezahlukeneyo zophando.

● Inkqubo engqongqo yolawulo lomgangatho: Iindawo zolawulo lomgangatho ongundoqo kuwo onke amanyathelo abandakanya ukulungiswa kwesampulu, ukulungiswa kwethala leencwadi, ulandelelwano kunye ne-bioinformatics ziphantsi kweliso elibukhali ukuze kunikezelwe iziphumo ezikumgangatho ophezulu.

● Oovimba beenkcukacha abaninzi abakhoyo ukuze bachaze umsebenzi kunye nezifundo zokutyebisa ukuzalisekisa iinjongo zophando ezahlukeneyo.

● Iinkonzo zokuthengisa emva kokuthengisa: Iinkonzo emva kokuthengiswa zivumelekile kwiinyanga ezi-3 ekugqityweni kweprojekthi, kubandakanywa ukulandelwa kweeprojekthi, ukuxazulula iingxaki, iziphumo ze-Q & A, njl.

IiMfuno zeSampulu kunye nokuhanjiswa

Ithala leencwadi	Isicwangciso sokulandelelana	Idatha iyacetyiswa	Ulawulo lwemeko
UPoly A utyebile	Illumina PE150	6Gb	Q30≥85%

IiMfuno zeSampulu:

IiNucleotides:

Conc.(ng/μl)

Imali (μg)

Ubunyulu

Ingqibelelo

≥ 20

≥ 0.5

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Unyino okanye akukho protein okanye ungcoliseko lweDNA olubonakaliswe kwijeli.

Kwizityalo: RIN≥6.5;

Kwizilwanyana: RIN≥7.0;

5.0≥28S/18S≥1.0;

umphakamo osisiseko okanye ongekhoyo

Izicubu: Ubunzima (bomileyo):≥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 kuno 5×105.

Iisampulu zegazi:Umthamo≥1 ml

I-Microorganism:Ubunzima ≥ 1 g

Unikezelo lweSampuli eCetyisiweyo

Isikhongozeli: 2 ml centrifuge ityhubhu (Ifoyile yeTin ayikhuthazwa)

Isampuli yokuleyibhela: Iqela+liphindaphinda umz. A1, A2, A3;B1, B2, B3... ...

Ukuthunyelwa ngenqanawa:

Umkhenkce owomileyo: Iisampulu 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.