Usoro mRNA dị ogologo-Nanopore
Uru ọrụ
● Eleghị anya usoro dị ala
● Na-ekpughe ụmụ irighiri ihe cDNA zuru oke
● Obere data achọrọ iji kpuchie otu ọnụọgụ ederede
● Ịmata ọtụtụ isoforms kwa mkpụrụ ndụ ihe nketa
● Ngosipụta nkwupụta na ọkwa isoform
Nkọwapụta ọrụ
| Ụlọ akwụkwọ | Platform | Mmụba data akwadoro (Gb) | Njikwa ogo |
| cDNA-PCR (Poly-A bara ụba) | Nanopore PromethION P48 | 6 Gb/ sample (dabere na ụdị) | Oke zuru oke - 70% Nkezi akara ogo: Q10
|
Nyocha bioinformatics
●Nhazi data raw
● njirimara transcript
● Nhọrọ ọzọ splicing
● Nkọwapụta n'ogo mkpụrụ ndụ ihe nketa na ọkwa isoform
● Nyocha okwu dị iche iche
● Nkọwa ọrụ na ịba ụba (DEGs na DETs)
Ihe nlele chọrọ na nnyefe
Ihe atụ chọrọ:
Nucleotides:
| Conc.(ng/μl) | Ọnụ ego (μg) | Ịdị ọcha | Iguzosi ike n'ezi ihe |
| ≥ 100 | ≥ 0.6 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Enwere oke ma ọ bụ enweghị protein ma ọ bụ mmetọ DNA nke egosiri na gel. | Maka osisi: RIN≥7.0; Maka anụmanụ: RIN≥7.5; 5.0≥28S/18S≥1.0; nwere oke ma ọ bụ enweghị mgbago elu |
Anụ ahụ: Ibu (akọrọ): ≥1 g
* Maka anụ ahụ dị obere karịa 5 mg, anyị na-akwado iziga ihe nlele anụ ahụ kpọnwụrụ akpọnwụ (na mmiri mmiri nitrogen).
Nkwụsị cell: Ọnụ ọgụgụ cell = 3×106- 1 × 107
* Anyị na-akwado ibupu cell lysate oyi kpọnwụrụ.Ọ bụrụ na sel ahụ riri obere karịa 5 × 105, Flash jụrụ oyi na mmiri mmiri nitrogen ka akwadoro, nke ka mma maka mmịpụta micro.
Ihe nlele ọbara: olu≥1 ml
Nnyefe nlele akwadoro
Akpa: 2 ml centrifuge tube (Tin foil adịghị akwadoro)
Ịkpọ aha nlele: Otu+ megharịa dịka A1, A2, A3;B1, B2, B3......
Mbupu: 2, akọrọ-ice: Sample mkpa ka a juru n'ọnụ na akpa na-eli ozu na akọrọ-ice.
- Ọkụ RNAstable: Enwere ike ịkpọsa sample RNA na tube stabilization RNA (dịka ọmụmaatụ RNAstable®) ma bufee ya n'ime ụlọ okpomọkụ.
Usoro ọrụ ọrụ
Nucleotides:
Nbufe ihe atụ
Ịrụ ụlọ akwụkwọ
Usoro
Nyocha data
Mgbe-ire ọrụ
Usoro ọrụ ọrụ
Anụ ahụ:
Nnwale imewe
Nbufe ihe atụ
mmịpụta RNA
Ịrụ ụlọ akwụkwọ
Usoro
Nyocha data
Mgbe-ire ọrụ
1.Nyocha okwu dị iche iche - nkata mgbawa ugwu
Enwere ike ịhazi nyocha okwu dị iche na ọkwa mkpụrụ ndụ abụọ iji chọpụta mkpụrụ ndụ ihe nketa egosipụtara dị iche iche (DEGs) yana ọkwa dị iche iche iji mata ihe dị iche.
ederede ederede (DET)
2.Map ikpokọta ọkwa dị elu
3.Alternative splicing njirimara na nhazi ọkwa
Astalavista nwere ike ibu amụma ụdị ise dị iche iche nke mmemme ịgbasa.
4.Nhọrọ poly-adenylation (APA) ihe omume njirimara na Motif na 50 bp elu nke poly-A
Okwu BMK
Nchọpụta splicing ọzọ na nha ọkwa isoform site na nanopore usoro transcriptome zuru oke.
Ebipụtara:Nkwukọrịta okike, 2020
Atụmatụ usoro:
Nchịkọta: 1. CLL-SF3B1 (WT);2. CLL-SF3B1 (K700E mutation);3. Nkịtị B-cell
Usoro usoro: MinION 2D usoro ọba akwụkwọ, Usoro usoro ọba akwụkwọ PromethION 1D;mkpiri-agụ data si n'otu sample
Usoro nhazi usoro: Nanopore MinION;Nanopore PromethION;
Nsonaazụ igodo
1.Isoform-level Alternative splicing Identification
Usoro ọgụgụ ogologo na-enye ike njirimara nke mutant SF3B1K700E-gbanwere saịtị splice na ọkwa isoform.Achọpụtara 35 ọzọ 3′SSs na 10 ọzọ 5′SSs dị iche iche n'etiti SF3B1K700Ena SF3B1WT.33 n'ime 35 mgbanwe ka achọpụtara ọhụrụ site n'usoro ogologo ọgụgụ.
2.Isoform-level Alternative Splicing quantification
Ngosipụta nke njide intron (IR) isoforms na SF3B1K700Ena SF3B1WTA na-agụta ya dabere na usoro nanopore, na-ekpughe usoro nchịkwa zuru ụwa ọnụ nke isoforms IR na SF3B1.K700E.
Ntụaka
Tang AD, Soulette CM, Baren MJV, et al.Nkọwa ederede zuru oke nke SF3B1 mutation na ọrịa leukemia lymphocytic na-adịghị ala ala na-ekpughe mbelata nke introns ejidere [J].Nkwukọrịta okike.
