Yakareba isiri-coding kutevedzana-Illumina
Basa Rinobatsira
● Zvakanakira Basa
● Serura nematishu chaiwo
● Danho chairo rinoratidza uye rinopa shanduko yekutaura ine simba
● Maitiro chaiwo enguva uye maratidziro emuchadenga
● Kuongororwa kwakabatana nemRNA data.
● BMKCloud-based result delivery: Customized data-mining inowanikwa pachikuva.
● Mushure mekutengesa masevhisi anoshanda kwemwedzi mitatu pakupedzwa kweprojekiti
Sample Zvinodiwa uye Delivery
| Library | Platform | Yakakurudzirwa data | Data QC |
| rRNA kupera | Illumina PE150 | 10 Gb | Q30≥85% |
| Conc.(ng/μl) | Mari (μg) | Kuchena | Kuperera |
| ≥ 100 | ≥ 0.5 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Yakaganhurirwa kana isina mapuroteni kana kusvibiswa kweDNA kunoratidzwa pagel. | Kumiti: RIN≥6.5; Zvemhuka: RIN≥7.0; 5.0≥28S/18S≥1.0; kukwirira kwakadzika kana kusina kwekutanga |
Nucleotides:
Matumbu: Huremu(yakaoma): ≥1 g
*Kune tishu diki pane 5 mg, tinokurudzira kutumira flash yakaomeswa nechando (mumvura nitrogen) sampuli yetishu.
Kumiswa kwesero: Kuwanda kwesero = 3 × 107
* Isu tinokurudzira kutumira chando cell lysate.Kana iyo sero inoverenga idiki pane 5 × 105, flash yakaomeswa munitrogen yemvura inokurudzirwa.
Mienzaniso yeropa:
PA×geneBloodRNATube;
6mLTRIzol uye 2mL ropa(TRIzol:Ropa=3:1)
Yakakurudzirwa Sample Delivery
Container: 2 ml centrifuge chubhu (Tin foil haina kukurudzirwa)
Sample labeling: Boka+ replicate eg A1, A2, A3;B1, B2, B3...
Shipment:
1.Dry-ice: Samples dzinoda kuiswa mumabhegi uye kuvigwa muoma-ice.
2.RNAstable tubes: RNA samples inogona kuomeswa muRNA stabilization tube (eg RNAstable®) uye inotumirwa mukupisa kwekamuri.
Kuyerera Kwebasa Rebasa
Kuedza kugadzira
Muenzaniso wekutumira
RNA kubviswa
Kuvakwa kweraibhurari
Sequencing
Data analysis
Mushure mekutengesa masevhisi
Bioinformatics
1.LncRNA classification
LncRNA yakafanotaurwa nemasoftware mana ari pamusoro akaiswa muzvikamu zvina: lincRNA, anti-sense-LncRNA, intronic-LncRNA;pfungwa-LncRNA.LncRNA classification yakaratidzwa mune histogram pazasi.
LncRNA classification
2.Cis-targeted genes yeDE-lncRNA enrichment analysis
ClusterProfiler aishandiswa muGO enrichment analysis pane cis-targeted genes of differentially expression lncRNA (DE-lncRNA), maererano nebiological process, molecular function and cell components.GO enrichment ongororo inzira yekuona DEG-inotungamirwa zvakanyanya kupfumisa GO mazwi kana ichienzaniswa neyese genome.Matemu akafumiswa akaiswa muhistogram, bubble chart, nezvimwewo sezvaratidzwa pazasi.
Cis-targeted genes yeDE-lncRNA enrichment analysis -Bubble chati
3. Nokuenzanisa kureba, nhamba yeexon, ORF uye uwandu hwekutaura kwemRNA nelncRNA, tinogona kunzwisisa kusiyana kwechimiro, kutevedzana uye zvichingodaro pakati pazvo, uyewo kuongorora kana novel lncRNA yakafanotaurwa nesu inopindirana nemaitiro akazara.
Nyaya yeBMK
Deregulated lncRNA expression profil mumouse lung adenocarcinomas ine KRAS-G12D mutation uye P53 knockout.
Rakatsikiswa:Nyaya yeSerura uye Molecular Medicine,2019
Sequencing strategy
Illumina
Sample collection
Iyo NONMMUT015812-knockdown KP (shRNA-2) masero uye maitiro asina kunaka (sh-Scr) masero akawanikwa pazuva rechitanhatu rehutachiona hwehutachiona.
Mibairo yakakosha
Chidzidzo ichi chinoongorora lncRNAs inoratidzwa zvisina tsarukano mumbeva lung adenocarcinoma ine P53 knockout uye KrasG12D mutation.
1.6424 lncRNAs yakaratidzwa zvakasiyana (≥ 2-fold change, P <0.05).
2.Pakati pese 210 lncRNAs (FC≥8), 11 lncRNAs 'kutaura kwakagadziriswa neP53, 33 lncRNAs neKRAS uye 13 lncRNAs ne hypoxia mumasero ekutanga eKP, maererano.
3.NONMMUT015812, iyo yaishamisa-yakagadziriswa mu mouse lung adenocarcinoma uye yakarongedzwa zvisina kunaka neP53 re-expression, yakaonekwa kuti iongorore basa rayo remagetsi.
4.Knockdown yeNONMMUT015812 ne shRNAs yakaderedza kuwanda uye kukwanisa kutama kwemasero eKP.NONMMUT015812 yaive mukana oncogene.
 KEGG nzira yekuongorora yemajini anoratidzwa zvakasiyana muNONMMUT015812-knockdown KP masero. |  Gene Ontology kuongororwa kwemajini anoratidzwa zvakasiyana muNONMMUT015812-knockdown KP masero. |
Reference
Deregulated lncRNA expression profile in the mouse lung adenocarcinomas ine KRAS-G12D mutation uye P53 knockout [J].Chinyorwa cheSerura uye Molecular Medicine, 2019, 23 (10).DOI: 10.1111/jcmm.14584
