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ọkọlọtọ-03

Ngwaahịa

Eukaryotic mRNA Sequencing-Ilumina

usoro mRNA na-enyere aka ịkọwapụta mRNA niile edere site na sel n'okpuru ọnọdụ akọwapụtara.Ọ bụ teknụzụ siri ike maka ikpughe profaịlụ mkpụrụ ndụ ihe nketa, usoro mkpụrụ ndụ ihe nketa na usoro mkpụrụ ndụ nke ụfọdụ usoro ndu.Ruo ugbu a, a na-arụ ọrụ mRNA usoro n'ọtụtụ ebe na nyocha dị mkpa, nchọpụta ụlọ ọgwụ, mmepe ọgwụ, wdg.

Platform: Illumina NovaSeq Platform


Nkọwa ọrụ

Bioinformatics

Nsonaazụ ngosi

Uru

● Ahụmahụ dị ukwuu: A na-ahazi ihe karịrị 200,000 samples na BMK na-ekpuchi ụdị ụdị dị iche iche, gụnyere omenala cell, anụ ahụ, mmiri ahụ, wdg na ihe karịrị 7,000 mRNA-Seq ọrụ mechiri kpuchiri mpaghara nyocha dị iche iche.

● Usoro njikwa mma siri ike: Isi njikwa njikwa mma site na usoro niile gụnyere nkwadebe nlele, nkwadebe ọbá akwụkwọ, usoro na bioinformatics ka nọ n'okpuru nlekọta anya iji wepụta nsonaazụ dị elu.

● Ọtụtụ ọdụ data dị maka nkọwa ọrụ na ọmụmụ ịba ụba iji mezuo ebumnuche nyocha dị iche iche.

● Ọrụ ire ere: Ọrụ ire ere na-arụ ọrụ maka ọnwa 3 mgbe arụchara ọrụ, gụnyere nleba anya ọrụ, mgbapụ nsogbu, nsonaazụ Q&A, wdg.

Ihe nlele chọrọ na nnyefe

Ụlọ akwụkwọ Usoro usoro akwadoro data Njikwa ogo
Poly A bara ụba Ilumina PE150

6 gb

Q30≥85%

Ihe atụ chọrọ:

Nucleotides:

Conc.(ng/μl)

Ọnụ ego (μg)

Ịdị ọcha

Iguzosi ike n'ezi ihe

≥ 20

≥ 0.5

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≥6.5;

Maka anụmanụ: RIN≥7.0;

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.

Ihe nlele ọbara:Olu ≥1 ml

Microorganism:Mass ≥ 1 g

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. Ikpụrụ kpọrọ nkụ: Ọ dị mkpa ka a kwakọba ihe nlele n'ime akpa wee lie ya na ice kpọrọ nkụ.
  2. Ọkụ RNAstable: Enwere ike ịkpọsa sample RNA na tube stabilization RNA (dịka ọmụmaatụ RNAstable®) ma bufee ya n'ime ụlọ okpomọkụ.

Usoro ọrụ ọrụ

Ihe atụ QC

Nnwale imewe

nnyefe sample

Nbufe ihe atụ

Nnwale pilot

mmịpụta RNA

Nkwadebe ụlọ akwụkwọ

Ịrụ ụlọ akwụkwọ

Usoro

Usoro

Nyocha data

Nyocha data

Mgbe ire Ọrụ

Mgbe-ire ọrụ


  • Nke gara aga:
  • Osote:

  • Bioinformatics

    wps_doc_10

    Eukaryotic mRNA usoro nyocha usoro ọrụ

    Bioinformatics

    ØNjikwa ogo data raw

    ØNtụaka genome alignment

    ØNyocha nhazi nke transcript

    ØNgosipụta okwu

    ØNyocha okwu dị iche iche

    ØNkọwa ọrụ na ịba ụba

    1.mRNA Data Saturation curve

    3 (1)

    2.Ntụle okwu dị iche - nkata mgbawa ugwu

    4(1)

    3.Nkọwa KEGG na DEGs

    5 (1)

    4.GO nhazi ọkwa na DEGs

    6 (1)

    nweta ntinye okwu

    Dee ozi gị ebe a ziga anyị ya

    Zitere anyị ozi gị: