Usoro mRNA dị ogologo-Nanopore
Atụmatụ
● Ijide poly-A mRNA sochiri cDNA njikọ na nkwadebe ọbá akwụkwọ
● Usoro nke ederede zuru oke
● Nyocha bioinformatic dabere na ntinye na genome ntụaka
● Nyocha bioinformatic na-agụnye ọ bụghị nanị okwu na mkpụrụ ndụ ihe nketa na ọkwa isoform kamakwa nyocha nke lncRNA, mkpụrụ ndụ ihe nketa, poly-adenylation na usoro mkpụrụ ndụ ihe nketa.
Uru ọrụ
●Nkọwapụta okwu na ọkwa isoform: na-enyere nyocha nkọwa zuru oke na nke ziri ezi, mkpughe mgbanwe nke enwere ike kpuchie mgbe ị na-enyocha mkpụrụ ndụ ihe nketa niile.
●Achọrọ data mbelata:Tụnyere usoro nke ọgbọ na-esote (NGS), usoro Nanopore na-egosipụta ihe achọrọ data dị ala, na-enye ohere maka nha nha nha nha njupụta mkpụrụ ndụ ihe nketa yana obere data.
●Izi ezi dị elu nke ngụpụta okwu: ma na gene na isoform larịị
●Nchọpụta ozi ntụgharị ederede agbakwunyere: polyadenylation ọzọ, mkpụrụ ndụ ihe nketa na lcnRNA na mkpụrụ ndụ ihe nketa ha
●Ọkachamara sara mbara: Ndị otu anyị na-eweta ahụmịhe bara ụba na ọrụ ọ bụla, ebe ha rụchara ọrụ transcriptome karịrị 850 Nanopore ma hazie ihe karịrị 8,000 sample.
●Nkwado maka ire ere: nkwa anyị ga-agafe karịa mmecha ọrụ yana oge ọrụ ọnwa 3 gachara. N'ime oge a, anyị na-enye nlebanya ọrụ, enyemaka nchọpụta nsogbu, yana oge ajụjụ & A iji zaa ajụjụ ọ bụla metụtara nsonaazụ ya.
Ihe nlele chọrọ na nnyefe
| Ụlọ akwụkwọ | Usoro usoro | akwadoro data | Njikwa ogo |
| Poly A bara ụba | Ilumina PE150 | 6/12 Gb | Nkezi akara ogo: Q10 |
Ihe atụ chọrọ:
Nucleotides:
| Conc.(ng/μl) | Ọnụ ego (μg) | Ịdị ọcha | Iguzosi ike n'ezi ihe |
| ≥ 100 | ≥ 1.0 | 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 |
● Osisi:
Mgbọrọgwụ, azuokokoosisi ma ọ bụ petal: 450 mg
Akwụkwọ ma ọ bụ mkpụrụ: 300 mg
Mkpụrụ: 1.2 g
● Anụmanụ:
Obi ma ọ bụ eriri afọ: 300 mg
Viscera ma ọ bụ ụbụrụ: 240 mg
Akwara: 450 mg
Ọkpụkpụ, ntutu ma ọ bụ akpụkpọ: 1g
● Arthropods:
Ụmụ ahụhụ: 6g
Crustacea: 300 mg
● Ọbara dum: 1 tube
● Selụ: 106 mkpụrụ ndụ
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:
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
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
● 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)
Ntụle splicing ọzọ
Nhọrọ Polyadenylation Analysis (APA)
amụma lncRNA
Nkọwa nke mkpụrụ ndụ ihe nketa
Nchịkọta nke DET
Netwọk Protein-Protein na DEGs
Nyochaa ọganihu nke BMKGene's Nanopore zuru ezu usoro mRNA na-akwado site na nchịkọta mbipụta.
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