ʻO ke kaʻina mRNA lōʻihi piha -PacBio
Pono lawelawe
● Heluhelu pololei i ka mole cDNA lōʻihi piha mai ka hopena 3'- a hiki i ka hopena 5'-
● Iso-form pae hoʻonā ma ke kaʻina hana
● Nā palapala me ka pololei kiʻekiʻe a me ka pololei
● kūpono loa i nā ʻano waiours
● Hiki ke hoʻopili nui me 4 PacBio Sequel II sequencing platform i lako
● Loaʻa i ka ʻike me ka ʻoi aku o 700 Pacbio-based RNA sequencing projects
● Hāʻawi i nā hualoaʻa ma muli o BMKCloud: Loaʻa ka ʻili ʻikepili i hana ʻia ma ka paepae.
● Pono nā lawelawe ma hope o ke kūʻai aku no 3 mahina ma hope o ka pau ʻana o ka papahana
Nā kikoʻī lawelawe
Papahana: PacBio Sequel II
Waihona waihona: Poly A- hoʻonaʻauao mRNA waihona
Hāʻawi ʻia ka ʻikepili i manaʻo ʻia: 20 Gb/lapana (Ma muli o nā ʻano ʻano)
FLNC（%）： ≥75%
*FLNC: Nā transcipts non-chimeric lōʻihi piha
Nā loiloi bioinformatics
● Hoʻoponopono ʻikepili maka
● ʻIke kope kope
● Hoʻonohonoho kaʻina
● Ka helu ʻana
● Palapala Hana
Nā Koina Laʻana a me ka Hoʻouna ʻana
Nā Koina Laʻana:
Nucleotides:
| Conc.(ng/μl) | Ka nui (μg) | Maemae | Pono pono |
| ≥ 120 | ≥ 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≥7.5; No nā holoholona: RIN≥8.0; 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 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
Microorganism:Ka nui ≥ 1 g
Manaʻo ʻia ka hāʻawi laʻana
ipu:
2 ml centrifuge paipu (ʻaʻole ʻōlelo ʻia ʻo Tin foil)
Laʻana lepili: Pūʻulu+hoʻopiʻi e laʻa me A1, A2, A3;B1, B2, B3... ...
Hoʻouna:
1. Ka hau maloo: Pono e hoʻopili ʻia nā laʻana i loko o nā ʻeke a kanu ʻia i loko o ka hau maloʻo.
2. Nā paipu RNAstable: Hiki ke hoʻomaloʻo ʻia nā laʻana RNA i loko o ka paipu hoʻopaʻa RNA (e laʻa me RNAstable®) a hoʻouna ʻia i loko o ka lumi wela.
Holo Hana Hana
Hoʻolālā hoʻokolohua
Hāʻawi laʻana
RNA unuhi
Kūkulu hale waihona puke
Ke kaʻina ʻana
ʻIkepili ʻikepili
Nā lawelawe ma hope o ke kūʻai aku
1.FLNC puunaue loihi
Hōʻike ka lōʻihi o ka heluhelu non-chimeric piha piha (FLNC) i ka lōʻihi o ka cDNA i ka hana hale waihona.He hōʻailona koʻikoʻi ka hāʻawi ʻana i ka lōʻihi o FLNC i ka loiloi ʻana i ka maikaʻi o ke kūkulu hale waihona.
Heluhelu ʻia ka lōʻihi o FLNC
2. Hoʻopau i ka māhele ORF māhele lōʻihi
Hoʻohana mākou i ka TransDecoder e wānana i nā ʻāpana coding protein a me nā kaʻina amino acid e hoʻohua ai i nā pūʻulu unigene, kahi i loaʻa i ka ʻike transcript ʻole-redundant i nā laʻana āpau.
Hoʻopiha piha i ka māhele ʻāina ORF
3.KEGG ala hoʻonui hoʻonaʻauao
Hiki ke ʻike ʻia nā transcripts like ʻole (DETs) ma o ka hoʻohālikelike ʻana i ka ʻikepili sequencing RNA e pili ana i ka NGS ma nā pūʻulu transcript lōʻihi i hana ʻia e ka ʻikepili sequencing PacBio.Hiki i kēia mau DET ke hana hou no ka nānā 'ana i nā hana like 'ole, no ka la'ana, ka ho'onui 'ana i ke ala KEGG.
ʻO ka hoʻonui ʻana i ke alahele DET KEGG -Dot plot
Hihia BMK
ʻO ka hoʻomohala hoʻomohala o ka Populus stem transcriptome
Paʻi ʻia: Mea Puke Biotechnology Journal, 2019
Hoʻolālā kaʻina hana:
ʻOhi laʻana:ʻāpana ʻāpana: piko, internode mua (IN1), internode ʻelua (IN2), internode ʻekolu (IN3), internode (IN4) a me internode (IN5) mai Nanlin895
NGS-sequence:Ua hui ʻia ʻo RNA o 15 mau kānaka i hoʻokahi laʻana olaola.ʻEkolu hoʻopiʻi koʻikoʻi o kēlā me kēia helu i hana ʻia no ke kaʻina NGS
TGS-sequence:Ua māhele ʻia nā ʻāpana kumu i ʻekolu ʻāpana, ʻo ia hoʻi ka piko, IN1-IN3 a me IN4-IN5.Ua hana ʻia kēlā me kēia māhele no ka hoʻonohonoho ʻana o PacBio me nā ʻano hale waihona puke ʻehā: 0-1 kb, 1-2 kb, 2-3 kb a me 3-10 kb.
Nā hopena koʻikoʻi
1. Ua ʻike ʻia he 87150 mau moʻolelo holoʻokoʻa, kahi i ʻike ʻia ai 2081 novel isoforms a me 62058 novel alternative spliced isoforms.
Ua ʻike ʻia ka 2.1187 lncRNA a me 356 fusion genes.
3. Mai ka ulu mua a hiki i ka ulu lua, 15838 i hōʻike ʻokoʻa ʻia nā transcripts mai 995 mau genes i hōʻike ʻokoʻa ʻia.Ma nā DEG a pau, he 1216 nā kumu kākau, ʻaʻole i hōʻike ʻia ka hapa nui o ia mau mea.
Ua hōʻike ʻia ka loiloi hoʻonui 4.GO i ke koʻikoʻi o ka hoʻokaʻawale cell a me ke kaʻina hana hoʻemi oxidation i ka ulu mua a me ke kula kiʻekiʻe.
Nā hanana splicing ʻokoʻa a me nā isoform like ʻole
ʻO ka nānā ʻana o WGCNA ma nā kumu transcription
Kuhikuhi
Chao Q, Gao ZF, Zhang D, et al.ʻO ka dynamics hoʻomohala o ka Populus stem transcriptome.Mea kanu Biotechnol J. 2019;17(1):206-219.doi:10.1111/pbi.12958
