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Usoro mRNA zuru oke -PacBio

Usoro mRNA ogologo zuru oke -PacBio Foto egosipụtara

De novousoro transcriptome zuru oke, nke a makwaara dị kaDe novoIso-Seq na-ewere uru nke PacBio sequencer n'ogologo ọgụgụ, nke na-eme ka usoro nke mkpụrụ ndụ cDNA dị ogologo na-enweghị nkwụsịtụ ọ bụla.Nke a na-ezere kpam kpam njehie ọ bụla ewepụtara na usoro mgbakọ transcript wee wulite unigene sets na mkpebi ọkwa ọkwa isoform.Usoro unigene a na-enye ozi mkpụrụ ndụ ihe nketa siri ike dị ka “genome ntụaka” na ọkwa transcriptome.Na mgbakwunye, ijikọ na data usoro usoro ọgbọ na-abịa, ọrụ a na-enye ike njupụta ziri ezi nke nkwupụta ọkwa isoform.

Platform: PacBio Sequel II

Ọbá akwụkwọ: SMRT mgbịrịgba ọba akwụkwọ

Kpọtụrụ anyị

Nkọwa ọrụ

Nsonaazụ ngosi

Ọmụmụ ihe

Uru ọrụ

● Gụpụta molekul cDNA zuru oke site na 3'- ngwụcha ruo 5'- njedebe.

● Mkpebi ọkwa nke iso-ụdị n'usoro usoro

● Ederede nwere oke ziri ezi na iguzosi ike n'ezi ihe

● Dakọtara nke ukwuu na ụdị vaiours

● Nnukwu ikike nhazi nke nwere 4 PacBio Sequel II usoro nhazi usoro nke a kwadebere

● Ahụmahụ nke ukwuu nwere ihe karịrị 700 Pacbio nke usoro usoro usoro usoro RNA

● Nnyefe sitere na BMKCloud: Ngwuputa data ahaziri iche dị na ikpo okwu.

● Ọrụ ire ere gachara ọnwa 3 ka arụchara ọrụ

Nkọwapụta ọrụ

Platform: PacBio Sequel II

Ọbá akwụkwọ nke usoro: Ọbá akwụkwọ mRNA nwere Poly A

Mmụba data akwadoro: 20 Gb/ sample (dabere na ụdị)

FLNC (%): ≥75%

*FLNC: Mpempe akwụkwọ na-abụghị chimeric ogologo zuru oke

Nyocha bioinformatics

● Nhazi data raw

● njirimara transcript

● Usoro usoro

● Ọnụ ọgụgụ okwu

● Nkọwa ọrụ

Ihe nlele chọrọ na nnyefe

Ihe atụ chọrọ:

Nucleotides:

Conc.(ng/μl)

Ọnụ ego (μg)

Ịdị ọcha

Iguzosi ike n'ezi ihe

≥ 120

≥ 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≥7.5;

Maka anụmanụ: RIN≥8.0;

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

Microorganism:Mass ≥ 1 g

Nnyefe nlele akwadoro

akpa:
2 ml nke centrifuge tube (anaghị akwado foil tin)
Ịkpọ aha nlele: Otu+ megharịa dịka A1, A2, A3;B1, B2, B3......

Mbupu:

1. Dry-ice: A ga-etinye ihe nlele n'ime akpa ma lie ya na ice-akọrọ.
2. tubes RNAstable: Enwere ike ịkpọsa ihe nlele RNA na tube stabilization RNA (dịka ọmụmaatụ RNAstable®) ma bufee ya n'ime ụlọ okpomọkụ.

Usoro ọrụ ọrụ

Nnwale imewe

Nbufe ihe atụ

mmịpụta RNA

Ịrụ ụlọ akwụkwọ

Usoro

Nyocha data

Mgbe-ire ọrụ

Nke gara aga: Eukaryotic mRNA Sequencing-Ilumina

Osote: Usoro mRNA dị ogologo-Nanopore

1.FLNC ogologo nkesa

Ogologo nke ọgụgụ na-abụghị chimeric (FLNC) zuru oke na-egosi ogologo cDNA n'iwu ụlọ ọba akwụkwọ.Nkesa ogologo FLNC bụ ihe ngosipụta dị oke mkpa n'ịtụle ogo owuwu ụlọ ọba akwụkwọ.

mRNA-FLNC-agụ-ogologo-nkesa

Nkesa ogologo FLNC gụrụ

2.Complete ORF mpaghara ogologo nkesa

Anyị na-eji TransDecoder ịkọ amụma mpaghara nzuzo protein yana usoro amino acid kwekọrọ iji mepụta usoro unigene, nke nwere ozi transcript zuru oke na nlere niile.

Nkesa ogologo mRNA-zuru ezu-ORF

Nkesa ogologo mpaghara ORF zuru ezu

3.KEGG ụzọ nkwalite nkwalite

Enwere ike ịmata transcript (DET) dị iche iche site na ịhazi data usoro RNA dabere na NGS na nhazi transcript zuru oke nke data usoro PacBio mepụtara.Enwere ike ịhazi DET ndị a maka nyocha ọrụ dị iche iche, dịka nyocha nkwalite ụzọ KEGG.

mRNA-DEG-KEGG-ụzọ-ịba ụba

DET KEGG ụzọ nkwalite -Ntụpọ nkata

Okwu BMK

Ọnọdụ mmepe nke Populus stem transcriptome

Ebipụtara: Akwụkwọ akụkọ ihe ọkụkụ nke biotechnology, 2019

Atụmatụ usoro:
Nchịkọta ihe atụ:mpaghara stem: apex, internode nke mbụ (IN1), internode nke abụọ (IN2), internode nke atọ (IN3), internode (IN4) na internode (IN5) sitere na Nanlin895
Usoro NGS:Achịkọtara RNA nke mmadụ 15 dị ka otu ihe nlele.A haziri ụdị mkpụrụ ndụ atọ nke isi ihe ọ bụla maka usoro NGS
Usoro TGS:E kewara mpaghara azuokokoosisi ụzọ atọ, ya bụ apex, IN1-IN3 na IN4-IN5.Ejiri ụdị ọba akwụkwọ anọ hazie mpaghara ọ bụla maka usoro PacBio: 0-1 kb, 1-2 kb, 2-3 kb na 3-10 kb.

Nsonaazụ igodo

1. Achọpụtara mkpokọta 87150 n'ụzọ zuru oke, nke, 2081 novel isoforms na 62058 novel alternative spliced isoforms ka achọpụtara.
Achọpụtara 2.1187 lncRNA na mkpụrụ ndụ ihe nketa 356.
3.Site na isi uto na uto nke abụọ, 15838 dị iche iche gosipụtara transcripts sitere na 995 dị iche iche gosipụtara mkpụrụ ndụ ihe nketa.N'ime DEGs niile, 1216 bụ ihe odide ederede, ọtụtụ n'ime ha ka akọpụtabeghị.
4.GO enrichment analysis kpughere mkpa nke cell nkewa na oxidation-mbelata usoro na isi na nke abụọ ibu.