Imikhiqizo

Ukulandelana kwe-mRNA yobude obugcwele-Nanopore

Ubude obugcwele be-mRNA Ukulandelanisa-Nanopore Isithombe Esifakiwe

Ukulandelana kwe-RNA kube ithuluzi eliyigugu lokuhlaziya okuphelele kwe-transcriptome.Ngokungangabazeki, ukulandelana kwendabuko kokufunda okufushane kuzuze intuthuko eminingi ebalulekile lapha.Noma kunjalo, imvamisa ihlangabezana nemikhawulo ekuhlonzweni kobude obugcwele be-isoform, ukulinganisa, ukuchema kwe-PCR.

Ukulandelana kwe-Nanopore kuzihlukanisa kwezinye izinkundla zokulandelana, ngokuthi ama-nucleotide afundwa ngokuqondile ngaphandle kokuhlanganiswa kwe-DNA futhi akhiqize ukufunda isikhathi eside emashumini ama-kilobase.Lokhu kunika amandla okulotshiweyo okufundwayo okuqondile okuwela ubude obugcwele kanye nokubhekana nezinselele ezifundweni zeleveli ye-isoform.

Inkundla:I-Nanopore PromethION

Umtapowolwazi:cDNA-PCR

Xhumana nathi

Imininingwane Yesevisi

Imiphumela yedemo

Ucwaningo lwesigameko

Izinzuzo Zesevisi

● Ukuchema kokulandelana okuphansi

● Ukwembula ama-molecule e-cDNA anobude obugcwele

● Idatha encane edingekayo ukuze kufakwe inombolo efanayo yokulotshiwe

● Ukukhonjwa kwama-isoform amaningi isakhi ngasinye

● Ukulinganisa kwenkulumo ezingeni le-isoform

Imininingwane Yesevisi

Umtapowolwazi

Inkundla

Isivuno sedatha esinconyiwe (Gb)

Ikhwalithi yokulawula

I-cDNA-PCR(I-Poly-A inothisiwe)

I-Nanopore PromethION P48

6 Gb/isampula (Kuya ngezinhlobo)

Isilinganiso sobude obugcwele＞70%

Isilinganiso sekhwalithi yesikolo: Q10

I-Bioinformatics ihlaziya

●Ukucubungula idatha eluhlaza

● Ukuhlonza umbhalo

● Okunye ukuhlanganisa

● Ukulinganisa kokuvezwa ezingeni lofuzo kanye nezinga le-isoform

● Ukuhlaziywa kwenkulumo okuhlukile

● Isichasiselo somsebenzi nokunothisa (ama-DEG nama-DET)

Izidingo Zesampula Nokulethwa

Izimfuneko zesampula:

Ama-Nucleotides:

Conc.(ng/μl)

Inani (μg)

Ubumsulwa

Ubuqotho

≥ 100

≥ 0.6

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≥7.0;

Okwezilwane: RIN≥7.5;

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: Isibalo samaseli = 3×10⁶- 1 × 10⁷

*Sincoma ukuthi uthumele i-frozen cell lysate.Esimeni lapho lelo seli liba lincane kuno-5×10⁵, i-flash efriziwe ku-nitrogen eyi-liquid iyanconywa, okungcono kakhulu ekukhishweni okuncane.

Amasampula egazi: Ivolumu≥1 ml

Ukulethwa Kwesampula Okunconyiwe

Isitsha: 2 ml centrifuge tube (Tin foil ayinconywa)

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

Ukuthunyelwa: 2, 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.

Ukugeleza Komsebenzi Wesevisi

Ama-Nucleotides:

Ukulethwa kwesampula

Ukwakhiwa komtapo wolwazi

Ukulandelanisa

Ukuhlaziywa kwedatha

Amasevisi angemuva kokuthengisa

Ukugeleza Komsebenzi Wesevisi

Izicubu:

Idizayini yokuhlola

Ukulethwa kwesampula

Ukukhishwa kwe-RNA

Ukwakhiwa komtapo wolwazi

Ukulandelanisa

Ukuhlaziywa kwedatha

Amasevisi angemuva kokuthengisa

Okwedlule: Ukulandelana kwe-mRNA yobude obugcwele -PacBio

Olandelayo: I-10x Genomics Visium Spatial Transcriptome

1.Ukuhlaziywa kwesisho esihlukile -Isakhiwo seVolcano

Ukuhlaziywa kwesisho esihlukile kungacutshungulwa kuzo zombili izinga lofuzo ukuze kukhonjwe izakhi zofuzo ezivezwa ngokuhlukile (ama-DEG) nakuleveli ye-isoform ukuze kukhonjwe ngokuhlukile.

3(1)

okulotshiweyo okuveziwe (DETs)

2.Imephu yokushisa yeqoqo le-Hierarchical

3.Alternative splicing ukuhlonza kanye ngezigaba

Izinhlobo ezinhlanu zezinye izehlakalo zokuhlanganisa zingabikezelwa yi-Astalavista.

4.Ukuhlonza imicimbi ye-Alternative poly-adenylation (APA) kanye ne-Motif ku-50 bp phezulu nomfula we-poly-A

Icala le-BMK

Ukuhlonza okuhlukile kokuhlanganisa kanye nokulinganisa kwezinga le-isoform ngokulandelana kwe-nanopore yobude obugcwele be-transcriptome

Kushicilelwe:Ezokuxhumana Ngemvelo, 2020

Isu lokulandelanisa:

Amaqembu: 1. CLL-SF3B1(WT);2. CLL-SF3B1(K700E ukuguqulwa);3. Ama-B-cell ajwayelekile

Isu lokulandelanisa: Ukulandelana kwelabhulali ye-Minion 2D, ukulandelana kwelabhulali ye-PromethION 1D;idatha yokufunda okufushane evela kumasampuli afanayo

Inkundla yokulandelanisa: I-Nanopore MinION;I-Nanopore PromethION;

Imiphumela esemqoka

1.I-Isoform-level Alternative Splicing Identification

Ukulandelana okufunde isikhathi eside kunika amandla ukukhonjwa kwe-SF3B1 eguquguqukayo^K700E-altered splice sites at isoform-level.Amanye ama-3′SS angu-35 kanye nama-5′SS angu-10 atholwe ehlukaniswe ngokuphawulekayo phakathi kwe-SF3B1^K700Ekanye ne-SF3B1^WT.Okungu-33 kwezingu-35 okushintshiwe kusanda kutholwa ngokulandelana okufundwe isikhathi eside.

2.Izinga le-Isoform-level Alternative Splicing quantification

Ukuvezwa kwe-intron retention(IR) isoforms ku-SF3B1^K700Ekanye ne-SF3B1^WTbalinganiswa ngokusekelwe ekulandeleni kwe-nanopore, okuveza ukulawulwa phansi komhlaba wonke kwama-isoforms e-IR ku-SF3B1^K700E.

Ireferensi

Tang AD , Soulette CM , Baren MJV , et al.Ukulingiswa kokulotshiweyo okugcwele kobude be-SF3B1 kuguquko lwe-lymphocytic leukemia engapheli kuveza ukulawulwa kwama-intron agciniwe[J].Ukuxhumana Kwemvelo.