Cikakkun Tsawon mRNA Sequencing-Nanopore
Amfanin Sabis
● Rashin son zuciya
● Bayyana cikakken tsawon kwayoyin cDNA
● Ƙananan bayanai da ake buƙata don rufe adadin rubuce-rubuce iri ɗaya
● Gane nau'ikan isoforms da yawa a kowace kwayar halitta
● Ƙididdigar magana a matakin isoform
Ƙayyadaddun Sabis
| Laburare | Dandalin | Abubuwan da aka ba da shawarar (Gb) | Kula da inganci |
| cDNA-PCR (wadatar Poly-A) | Nanopore PromethION P48 | 6 Gb/samfuri (Ya danganta da nau'in) | Rago mai cikakken tsayi: 70% Matsakaicin ƙimar inganci: Q10
|
Binciken bioinformatics
●Raw data sarrafa
● Ƙirar rubutu
● Madadin splicing
● Ƙididdigar ƙididdigewa a matakin kwayoyin halitta da matakin isoform
● Nazarin magana daban-daban
● Bayanin Aiki da haɓakawa (DEGs da DETs)
Samfuran Bukatun da Bayarwa
Samfuran Bukatun:
Nucleotides:
| Conc.(ng/μl) | Adadin (μg) | Tsafta | Mutunci |
| ≥ 100 | ≥ 0.6 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Iyakance ko babu furotin ko gurɓataccen DNA da aka nuna akan gel. | Don tsire-tsire: RIN≥7.0; Na dabbobi: RIN≥7.5; 5.0≥28S/18S≥1.0; iyakance ko babu hawan tushe |
Nama: Nauyi (bushe): ≥1 g
* Don nama ƙasa da 5 MG, muna ba da shawarar aika samfurin nama daskararre (a cikin ruwa nitrogen).
Dakatar da salula: Ƙididdiga ta salula = 3×106- 1 × 107
*Muna ba da shawarar jigilar daskararrun cell lysate.Idan wannan tantanin halitta ya ƙidaya ƙasa da 5 × 105, walƙiya daskararre a cikin ruwa nitrogen ana ba da shawarar, wanda ya fi dacewa don hakar micro.
Samfuran jini: girma≥1 ml
Isar da Samfurin Nasiha
Kwantena: 2 ml bututu centrifuge (Ba a ba da shawarar foil tin ba)
Alamar samfur: Ƙungiya+ kwafi misali A1, A2, A3;B1, B2, B3......
Shipping: 2, Dry-kankara: Samfurori suna buƙatar a tattara su a cikin jaka kuma a binne su a bushe-kankara.
- RNAstable tubes: Ana iya bushe samfuran RNA a bututun daidaitawar RNA (misali RNAstable®) kuma a tura su cikin zafin jiki.
Gudun Aikin Sabis
Nucleotides:
Samfurin bayarwa
Gina ɗakin karatu
Jeri
Binciken bayanai
Bayan-sayar da sabis
Gudun Aikin Sabis
Nama:
Gwajin ƙira
Samfurin bayarwa
RNA cirewa
Gina ɗakin karatu
Jeri
Binciken bayanai
Bayan-sayar da sabis
1.Bambance-bambancen magana bincike - Ƙarfin wutar lantarki
Za'a iya sarrafa nazarin maganganun bambance-bambance a cikin matakin jinsin biyu don gano nau'ikan da aka bayyana daban-daban (DEGs) da kuma a matakin isoform don gano bambancin.
rubuce-rubucen da aka bayyana (DETs)
2.Taswirar taswirar tari mai tsari
3.Alternative splicing ganewa da rarrabuwa
Nau'o'i biyar na madadin abubuwan da suka faru za a iya tsinkaya ta Astalavista.
4.Alternative poly-adenylation (APA) gano abubuwan da suka faru da Motif a 50 bp sama na poly-A
BMK Case
Madadin tantancewa da ƙididdige matakin isoform ta hanyar nanopore cikakken tsawon kwafin jeri.
Buga:Sadarwar yanayi, 2020
Dabarun tsarawa:
Rukuni: 1. CLL-SF3B1(WT);2. CLL-SF3B1 (K700E maye gurbi);3. Kwayoyin B na al'ada
Dabarun jeri: MinION 2D jerin laburare, Tsarin laburare na PromethION 1D;gajeriyar karanta bayanai daga samfurori iri ɗaya
Tsarin tsari: Nanopore MinION;Nanopore PromethION;
Sakamako mai mahimmanci
1.Isoform-matakin Alternative Slicing Identification
Jerin dogon karantawa yana ba da ikon gano mutant SF3B1K700E-canza shafukan yanar gizo a matakin isoform.35 madadin 3'SSs da 10 madadin 5'SSs an gano suna da banbanci sosai tsakanin SF3B1K700Ekuma SF3B1WT.33 daga cikin 35 canje-canje an gano su ta hanyar jerin dogon karatu.
2.Isoform-level Alternative Slicing quantification
Bayanin riƙewar intron (IR) isoforms a cikin SF3B1K700Ekuma SF3B1WTAn ƙididdige su bisa ga jerin nanopore, yana nuna ƙa'idodin ƙa'idodin duniya na IR isoforms a cikin SF3B1K700E.
Magana
Tang AD , Soulette CM , Baren MJV , et al.Cikakkun bayanan kwafi na SF3B1 maye gurbi a cikin cutar sankarar lymphocytic na yau da kullun yana bayyana raguwar abubuwan da aka riƙe [J].Sadarwar yanayi.
