Bolelele bo Feletseng ba mRNA Sequencing-Nanopore
Likaroloana
● Ho nka poly-A mRNA e lateloang ke cDNA synthesis le ho lokisa laebrari
● Tatellano ea likopi tse bolelele bo felletseng
● Tlhahlobo ea bioinformatic e thehiloeng ho ikamahanya le genome ea litšupiso
● Tlhahlobo ea bioinformatiki ha e akarelletse feela polelo ho liphatsa tsa lefutso le boemo ba isoform empa hape le tlhahlobo ea lncRNA, ho kopanngoa ha liphatsa tsa lefutso, poly-adenylation le gene structure.
Melemo ea Tšebeletso
●Quantification ea polelo boemong ba isoform: ho nolofalletsa tlhahlobo e qaqileng le e nepahetseng ea polelo, e senola phetoho e ka 'nang ea patoa ha ho hlahlojoa poleloana eohle ea lefutso.
●Litlhoko tsa Lintlha tse Fokotsoeng:Ha ho bapisoa le Next-Generation Sequencing (NGS), Nanopore sequencing e bonts'a litlhoko tse tlase tsa data, e lumellang maemo a lekanang a gene expression quantification saturation le data e nyane.
●Ho nepahala ho hoholo ha polelo ea quantification: ka bobeli boemong ba lefutso le isoform
●Ho tsebahatsa lintlha tse eketsehileng tsa transcriptomic: mefuta e meng ea polyadenylation, liphatsa tsa lefutso tsa fusion le lcnRNA le liphatsa tsa bona tsa lefutso
●Tsebo e Pharalletseng: Sehlopha sa rona se tlisa boiphihlelo bo bongata morerong o mong le o mong, kaha se qetile merero e fetang 850 ea Nanopore ea bolelele bo felletseng mme se sebelitse lisampole tse fetang 8,000.
●Tšehetso ea Post-Sales: Boitlamo ba rona bo fetela ka nģ'ane ho ho phethoa ha morero ka nako ea tšebeletso ea likhoeli tse 3 ka mor'a thekiso. Nakong ena, re fana ka tatellano ea projeke, thuso ea ho rarolla mathata, le linako tsa Q&A ho araba lipotso life kapa life tse amanang le sephetho.
Litlhoko tsa Mohlala le Phano
| Laebrari | Leano la tatellano | Lintlha li khothalelitsoe | Taolo ea Boleng |
| Poly A e atlehile | Illumina PE150 | 6/12 Gb | Karolelano ea lintlha tsa boleng: Q10 |
Litlhoko tsa Mohlala:
Li-nucleotides:
| Conc.(ng/μl) | Chelete (μg) | Bohloeki | Botšepehi |
| ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Tšoaetso e fokolang kapa ha e na protheine kapa DNA e bontšitsoeng ho gel. | Bakeng sa limela: RIN≥7.0; Bakeng sa liphoofolo: RIN≥7.5; 5.0≥28S/18S≥1.0; bophahamo bo fokolang kapa bo se nang motheo |
● Limela:
Motso, Stem kapa Petal: 450 mg
Lekhasi kapa Peō: 300 mg
Litholoana: 1.2 g
● Phoofolo:
Pelo kapa mala: 300 mg
Viscera kapa Boko: 240 mg
mesifa: 450 mg
Masapo, Moriri kapa Letlalo: 1g
● Arthropods:
Likokoanyana: 6g
Crustacea: 300 mg
● Mali a feletseng:1 tube
● Lisele: 106 lisele
Tlhahiso ea Mohlala e khothalelitsoeng
Setshelo: 2 ml centrifuge tube (Tin foil ha e khothalletsoe)
Mohlala oa ho ngola: Sehlopha+katiso mohlala A1, A2, A3; B1, B2, B3.
Thomello:
1. Leqhoa le omileng: lisampole li lokela ho kenngoa ka mekotleng ebe li patoa ka leqhoa le omileng.
2. Li-tubes tsa RNAstable: lisampole tsa RNA li ka omisoa ka har'a tube ea ho tsitsisa ea RNA (mohlala, RNAstable®) le ho romelloa ka mocheso oa kamore.
Phallo ea Mosebetsi oa Ts'ebeletso
Li-nucleotides:
Mohlala oa thomello
Kaho ea laebrari
Tatelano
Tlhahlobo ea lintlha
Litšebeletso tsa ka mor'a thekiso
Phallo ea Mosebetsi oa Ts'ebeletso
Tishu:
Moqapi oa liteko
Mohlala oa thomello
Tlhahiso ea RNA
Kaho ea laebrari
Tatelano
Tlhahlobo ea lintlha
Litšebeletso tsa ka mor'a thekiso
● Ts'ebetso ea data e tala
● Boitsebiso ba sengoloa
● Mokhoa o mong oa ho kopanya
● Tlhaloso ea polelo boemong ba liphatsa tsa lefutso le boemo ba isoform
● Tlhahlobo e fapaneng ea polelo
● Tlhaloso ea tšebetso le ntlafatso (DEGs le DETs)
Alternative splicing analysis
Alternative Polyadenylation Analysis (APA)
Boprofeta ba lncRNA
Tlhaloso ea liphatsa tsa lefutso tse ncha
Ho kopanya ha DETs
Protein-Protein Networks ho li-DEGs
Lekola tsoelopele e tsamaisoang ke BMKGene's Nanopore's Nanopore ea bolelele bo felletseng ba lits'ebeletso tsa tatelano ea mRNA ka pokello e hlophisitsoeng ea lingoliloeng.
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Eena, Z. et al. (2023) 'Tlhaloso ea nako e telele ea transcriptome ea li-lymphocyte e arabela IFN-γ e senola karabelo ea 'mele ea ho itšireletsa mafung ea Th1 ka flounder (Paralichthys olivaceus)', Fish & Shellfish Immunology, 134, leq. 108636. doi: 10.1016/J.FSI.2023.108636.
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Yu, D. et al. (2023) 'Nano-seq analysis e senola mekhoa e fapaneng ea ts'ebetso pakeng tsa li-exosomes le microvesicles tse nkiloeng ho hUMSC', Stem Cell Research le Therapy, 14 (1), maq. 1-13. doi: 10.1186/S13287-023-03491-5/TABLES/6.
