ʻO ka lōʻihi mRNA Sequencing-Nanopore
Pono lawelawe
● ʻO ke kaʻina hana haʻahaʻa
● Hōʻike i nā molekala cDNA piha
● ʻOi aku ka liʻiliʻi o ka ʻikepili no ka uhi ʻana i ka helu like o nā kope
● Ka ʻike ʻana o nā isoform he nui no kēlā me kēia gene
● Ka helu hōʻike ma ka pae isoform
Nā kikoʻī lawelawe
| Hale Waihona Puke | Papahana | Hāʻawi ʻia ka ʻikepili i manaʻo ʻia (Gb) | Ka Mana Mana |
| cDNA-PCR(Poly-A hoʻonui ʻia) | Nanopore PromethION P48 | 6 Gb/lapana (Ma muli o nā ʻano ʻano) | Lakiō piha piha＞70% Ka helu o ka maikaʻi maʻamau: Q10
|
Nā loiloi bioinformatics
●ʻO ka hana ʻikepili maka
● ʻIke kope kope
● Hoʻopili ʻokoʻa
● Ka helu hōʻike ma ka pae gene a me ka pae isoform
● Ka nānā 'ana i ka mana'o like 'ole
● Hōʻike a me ka hoʻonui ʻana i ka hana (DEG a me DET)
Nā Koina Laʻana a me ka Hoʻouna ʻana
Nā Koina Laʻana:
Nucleotides:
| Conc.(ng/μl) | Ka nui (μg) | Maemae | Pono pono |
| ≥ 100 | ≥ 0.6 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Hoʻopaʻa ʻia a ʻaʻole hoʻi i ka protein a i ʻole DNA contamination i hōʻike ʻia ma ka gel. | No nā mea kanu: RIN≥7.0; No nā holoholona: RIN≥7.5; 5.0≥28S/18S≥1.0; palena a ʻaʻole kiʻekiʻe kumu |
Tissue: Kaumaha (maloʻo): ≥1 g
*No ka ʻiʻo liʻiliʻi ma mua o 5 mg, manaʻo mākou e hoʻouna i kahi hāpana kikoo uila (i loko o ka nitrogen wai).
Hoʻokuʻu ʻia ke kelepona: helu kelepona = 3×106- 1×107
* Manaʻo mākou e hoʻouna i ka lysate cell frozen.Inā ʻoi aku ka liʻiliʻi o kēlā cell ma mua o 5 × 105, ʻōlelo ʻia ka flash frozen i loko o ka nitrogen wai, ʻoi aku ka maikaʻi no ka micro extraction.
Nā laʻana koko: Volume≥1 ml
Manaʻo ʻia ka hāʻawi laʻana
ʻO ka pahu: 2 ml centrifuge tube (ʻAʻole ʻōlelo ʻia ʻo Tin foil)
Laʻana lepili: Pūʻulu+hoʻopiʻi e laʻa me A1, A2, A3;B1, B2, B3... ...
Hoʻouna: 2、Dry-hau: Pono e hoʻopili ʻia nā laʻana i loko o nā ʻeke a kanu ʻia i loko o ka hau maloʻo.
- Nā paipu RNAstable: Hiki ke hoʻomaloʻo ʻia nā laʻana RNA i ka paipu hoʻopaʻa RNA (e laʻa me RNAstable®) a hoʻouna ʻia i loko o ka wela lumi.
Holo Hana Hana
Nucleotides:
Hāʻawi laʻana
Kūkulu hale waihona puke
Ke kaʻina ʻana
ʻIkepili ʻikepili
Nā lawelawe ma hope o ke kūʻai aku
Holo Hana Hana
ʻili:
Hoʻolālā hoʻokolohua
Hāʻawi laʻana
RNA unuhi
Kūkulu hale waihona puke
Ke kaʻina ʻana
ʻIkepili ʻikepili
Nā lawelawe ma hope o ke kūʻai aku
1.Ka nānā 'ana i ka mana'o like 'ole -Pāpala pele
Hiki ke hana ʻia ka ʻike ʻike ʻokoʻa ma ka pae gene no ka ʻike ʻana i nā genes i hōʻike ʻia ʻokoʻa (DEGs) a ma ka pae isoform e ʻike i ka ʻokoʻa.
nā palapala i hōʻike ʻia (DETs)
2.Palapala wela huikau hierarchical
3. Alternative splicing identification and classification
Hiki ke wānana ʻia e Astalavista ʻelima mau ʻano hanana splicing ʻē aʻe.
4.ʻIke ʻia nā hanana poly-adenylation (APA) a me ka Motif ma 50 bp i uka o ka poly-A
Hihia BMK
ʻO ka ʻike hoʻopili ʻokoʻa a me ka helu isoform-level e ka nanopore piha transcriptome sequencing.
Paʻi ʻia:Nature Communications, 2020
Hoʻolālā kaʻina hana:
Hui pū: 1. CLL-SF3B1(WT);2. CLL-SF3B1(K700E hoʻololi);3. Nā pūnaewele B maʻamau
Ka hoʻolālā hoʻonohonoho ʻana: MiniON 2D sequencing waihona, PromethION 1D sequencing hale waihona puke;ʻikepili heluhelu pōkole mai nā laʻana like
Papahana hoʻonohonoho: Nanopore Minion;Nanopore PromethION;
Nā hopena koʻikoʻi
1.Isoform-level Alternative Splicing Identification
Hāʻawi nā kaʻina heluhelu lōʻihi i ka ʻike ʻana i ka mutant SF3B1K700E- hoʻololi i nā pūnaewele splice ma ka pae isoform.Ua ʻike ʻia he 35 mau 3′SSs ʻokoʻa a me 10 mau 5′SSs ʻokoʻa ʻē aʻe ma waena o SF3B1.K700Ea me SF3B1WT.ʻO 33 o nā hoʻololi 35 i ʻike hou ʻia e nā kaʻina heluhelu lōʻihi.
2.Isoform-level Alternative Splicing quantification
Hōʻike o ka intron paʻa (IR) isoforms ma SF3B1K700Ea me SF3B1WTua helu ʻia ma muli o nā kaʻina nanopore, e hōʻike ana i kahi hoʻoponopono hoʻohaʻahaʻa honua o IR isoforms ma SF3B1K700E.
Kuhikuhi
ʻO Tang AD, Soulette CM, Baren MJV, a me al.Hōʻike ka hōʻike ʻana i ka transcript piha piha o ka mutation SF3B1 i ka maʻi maʻi lymphocytic maʻi i ka hoʻohaʻahaʻa ʻana o nā introns paʻa [J].Nature Communications.
