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Single-nuclei RNA Sequencing
Nts'etsopele ea ho ts'oaroa ha sele e le 'ngoe le mekhoa ea kaho ea laeborari e tloahelehileng, hammoho le tatellano ea maemo a holimo, e fetotse lithuto tsa liphatsa tsa lefutso maemong a lisele. Katleho ena e lumella tlhahlobo e tebileng le e felletseng ea lisele tse rarahaneng tsa lisele, ho hlola mefokolo e amanang le karolelano ea liphatsa tsa lefutso holim'a lisele tsohle le ho boloka ho fapana ha 'nete ka har'a batho bana. Le ha tatellano ea sele e le 'ngoe ea RNA (scRNA-seq) e na le melemo e ke keng ea latoloa, e kopana le liqholotso meleng e itseng moo ho theoang ho emisoa ha sele e le 'ngoe ho ipakang ho le thata mme ho hloka lisampole tse ncha. Ho BMKGene, re rarolla bothata bona ka ho fana ka tatellano ea "nucleus RNA" e le 'ngoe (snRNA-seq) re sebelisa theknoloji ea morao-rao ea 10X Genomics Chromium. Mokhoa ona o pharalatsa mefuta e mengata ea lisampole tse khonang ho hlahloba transcriptome boemong ba sele e le 'ngoe.
Ho aroloa ha li-nuclei ho finyelloa ka chip ea 10X Genomics Chromium e ncha, e nang le tsamaiso ea microfluidics e nang le mechine e robeli e nang le litsela tse peli. Ka har'a tsamaiso ena, lifaha tsa li-gel tse kenyelletsang li-barcode, li-primers, li-enzyme, le nucleus e le 'ngoe li kenngoa ka marotholi a oli a boholo ba nanoliter, ho etsa Gel Bead-in-Emulsion (GEM). Ka mor'a ho thehoa ha GEM, lisele tsa lysis le ho lokolloa ha barcode ho etsahala ka har'a GEM ka 'ngoe. Ka mor'a moo, limolek'hule tsa mRNA li fetoleloa ka morao ho li-cDNA, tse kenyelletsang li-barcode tsa 10X le Unique Molecular Identifiers (UMIs). Li-cDNA tsena li beoa tlas'a kaho e tloaelehileng ea tatellano ea laeborari, ho thusa tlhahlobo e matla le e felletseng ea liphatlalatso tsa liphatsa tsa lefutso boemong ba sele e le 'ngoe.
Sethala: 10× Genomics Chromium le Illumina NovaSeq Platform
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10x Genomics Visium Spatial Transcriptome
Spatial transcriptomics ke theknoloji e tsoetseng pele e lumellang bafuputsi ho etsa lipatlisiso tsa mefuta ea liphatsa tsa lefutso ka har'a lisele ha ba ntse ba boloka maemo a bona a sebaka. Sethala se le seng se matla sebakeng sena ke 10x Genomics Visium e kopantsoeng le tatellano ea Illumina. Molao-motheo oa 10X Visium o holim'a chip e khethehileng e nang le sebaka se khethiloeng sa ho hapa moo likarolo tsa lisele li behoang teng. Sebaka sena sa ho ts'oaroa se na le libaka tse nang le barcode, e 'ngoe le e' ngoe e tsamaellana le sebaka se ikhethileng ka har'a thishu. Limolek'hule tsa RNA tse hapuoeng ho tsoa liseleng ebe li ngotsoe ka li-identifiers tse ikhethang tsa limolek'hule (UMIs) nakong ea ts'ebetso ea ho fetolela ka morao. Libaka tsena tse nang le li-barcode le li-UMI li thusa ho etsa 'mapa o nepahetseng oa sebaka le ho lekanya liphatsa tsa lefutso ka qeto ea sele e le 'ngoe. Motsoako oa disampole tse nang le barcoded tsa sebaka le li-UMI li netefatsa ho nepahala le ho nepahala ha data e hlahisitsoeng. Ka ho sebelisa theknoloji ena ea Spatial Transcriptomics, bafuputsi ba ka fumana kutloisiso e tebileng ea mokhatlo oa sebaka sa lisele le litšebelisano tse rarahaneng tsa limolek'hule tse hlahang ka har'a lisele, tse fanang ka kutloisiso ea bohlokoa ka mekhoa ea mekhoa ea likokoana-hloko likarolong tse ngata, ho akarelletsa le oncology, neuroscience, tsoelo-pele ea biology, immunology. , le lithuto tsa botanical.
Sethala: 10X Genomics Visium le Illumina NovaSeq
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Bolelele bo Feletseng ba mRNA Sequencing-Nanopore
Le ha tatellano ea mRNA e thehiloeng ho NGS e le sesebelisoa se feto-fetohang sa ho lekanya liphatsa tsa lefutso, ho ts'epa ha eona lipalo tse khutšoane ho thibela katleho ea eona litlhahlobong tse rarahaneng tsa transcriptomic. Ka lehlakoreng le leng, tatellano ea nanopore e sebelisa theknoloji e baloang nako e telele, e nolofalletsang tatellano ea lingoliloeng tsa bolelele bo felletseng ba mRNA. Mokhoa ona o thusa ho hlahlobisisa ka botlalo mekhoa e meng ea ho arola, ho kopanya liphatsa tsa lefutso, poly-adenylation, le palo ea li-isoform tsa mRNA.
Nanopore sequencing, mokhoa o itšetlehileng ka nanopore-molek'hule e le 'ngoe ea motlakase oa motlakase oa nako ea sebele, e fana ka liphello tsa nako ea sebele. E tataisoa ke liprotheine tsa enjene, DNA e nang le likhoele tse peli e hokahana le liprotheine tsa nanopore tse kentsoeng ka har'a biofilm, e phutholoha ha e feta mocha oa nanopore tlasa phapang ea voltage. Lipontšo tse ikhethang tsa motlakase tse hlahisoang ke metheo e fapaneng holim'a DNA strand li fumanoa 'me li aroloa ka nako ea sebele, ho thusa ho latellana ho nepahetseng le ho tsoelang pele ha nucleotide. Mokhoa ona o mocha o hlōla mefokolo ea ho bala nako e khutšoanyane 'me o fana ka sethala se matla sa tlhahlobo e rarahaneng ea genomic, ho kenyelletsa le lithuto tse rarahaneng tsa transcriptomic, tse nang le liphello tsa hang-hang.
Sethala: Nanopore PromethION 48
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Tatelano ya bolelele bo felletseng ba mRNA -PacBio
Le ha tatellano ea mRNA e thehiloeng ho NGS e le sesebelisoa se feto-fetohang sa ho lekanya liphatsa tsa lefutso, ho itšetleha ha eona ka lipalo tse khutšoane ho thibela ts'ebeliso ea eona litlhahlobong tse rarahaneng tsa transcriptomic. Ka lehlakoreng le leng, PacBio sequencing (Iso-Seq) e sebelisa theknoloji e baloang nako e telele, e nolofalletsang tatellano ea lingoloa tsa bolelele bo felletseng ba mRNA. Mokhoa ona o thusa ho hlahloba ka botlalo mekhoa e meng ea ho arola, ho kopanya liphatsa tsa lefutso le poly-adenylation. Leha ho le joalo, ho na le likhetho tse ling tsa gene expression quantification ka lebaka la bongata bo bongata ba data bo hlokahalang. Theknoloji ea tatellano ea PacBio e itšetlehile ka tatellano ea molek'hule e le 'ngoe, nako ea sebele (SMRT), e fanang ka monyetla o ikhethang oa ho hapa litlaleho tsa bolelele bo feletseng ba mRNA. Mokhoa ona o mocha o kenyelletsa ho sebelisa li-zero-mode waveguides (ZMWs) le liliba tse entsoeng ka microfabricated tse nolofalletsang ho shebella nako ea sebele ea mosebetsi oa DNA polymerase nakong ea tatellano. Ka har'a li-ZMW tsena, PacBio's DNA polymerase e kopanya DNA e tlatselletsang, 'me e hlahisa lipalo tse telele tse nkang mongolo o felletseng oa mRNA. Ts'ebetso ea PacBio ka mokhoa oa Circular Consensus sequencing (CCS) e ntlafatsa ho nepahala ka ho latellana khafetsa molek'hule e tšoanang. Libalo tsa HiFi tse hlahisitsoeng li na le ho nepahala ho ka bapisoang le NGS, tse tlatsetsang hape tlhahlobong e felletseng le e tšepahalang ea likarolo tse rarahaneng tsa transcriptomic.
Sethala: PacBio Sequel II; PacBio Revio
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Eukaryotic mRNA Sequencing-NGS
Tatelano ea mRNA, theknoloji e feto-fetohang, e matlafatsa profiling e felletseng ea lingoliloeng tsohle tsa mRNA ka har'a lisele tlas'a maemo a ikhethileng. Ka ts'ebeliso ea eona e pharalletseng, sesebelisoa sena sa morao-rao se senola liphatlalatso tse rarahaneng tsa liphatsa tsa lefutso, libopeho tsa liphatsa tsa lefutso, le mekhoa ea limolek'hule e amanang le lits'ebetso tse fapaneng tsa tlhaho. E amohetsoe haholo lipatlisisong tsa mantlha, tlhahlobo ea bongaka, le nts'etsopele ea lithethefatsi, tatellano ea mRNA e fana ka leseli mabapi le ho rarahana ha matla a cellular le taolo ea liphatsa tsa lefutso, e tsosang bohelehele mabapi le bokhoni ba eona mafapheng a fapaneng.
Sethala: Illumina NovaSeq X; DNBSEQ-T7
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Non-Reference based mRNA Sequencing-NGS
tatellano ea mRNA e matlafatsa profil e felletseng ea lingoliloeng tsohle tsa mRNA ka har'a lisele tlas'a maemo a ikhethileng. Theknoloji ena e tsoetseng pele e sebetsa e le sesebelisoa se matla, se senolang liphatlalatso tse rarahaneng tsa liphatsa tsa lefutso, libopeho tsa liphatsa tsa lefutso, le mekhoa ea limolek'hule e amanang le mekhoa e fapaneng ea tlhaho. E amohetsoe haholo lipatlisisong tsa mantlha, tlhahlobo ea bongaka, le nts'etsopele ea lithethefatsi, tatellano ea mRNA e fana ka leseli mabapi le ho rarahana ha matla a cellular le taolo ea liphatsa tsa lefutso.
Sethala: Illumina NovaSeq X; DNBSEQ-T7
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Nako e telele e sa ngodiseng Sequencing-Illumina
Li-RNA tse telele tse sa khoutang (lncRNAs) li telele ho feta li-nucleotide tse 200 tse nang le bokhoni bo fokolang ba ho khouta ebile e le likarolo tsa bohlokoa ka har'a RNA e sa khouteng. E fumaneha nucleus le cytoplasm, li-RNA tsena li bapala karolo ea bohlokoa taolong ea epigenetic, transcriptal, le post-transcriptal, e totobatsang bohlokoa ba tsona ho bopeng lits'ebetso tsa cellular le limolek'hule. LncRNA sequencing ke sesebelisoa se matla ho arola lisele, Ontogenesis, le mafu a Batho.
Sethala: Illumina NovaSeq
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Nyenyane ea RNA Sequencing-Illumina
Limolek'hule tse nyenyane tsa RNA (sRNA), li kenyelletsa li-microRNAs (miRNAs), li-RNA tse nyenyane tse sitisang (siRNAs), le li-RNA tse sebetsanang le piwi (piRNAs). Har'a tsena, li-miRNA, tse ka bang bolelele ba 18-25 nucleotide, li bohlokoa haholo bakeng sa likarolo tsa tsona tsa bohlokoa tsa taolo lits'ebetsong tse fapaneng tsa cellular. Ka mekhoa e ikhethileng ea lisele le sethala, miRNA e bonts'a paballo e phahameng ho pholletsa le mefuta e fapaneng.
Sethala: Illumina NovaSeq
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CircRNA Sequencing-Illumina
Circry RNA sequencing (circRNA-seq) ke ho hlahisa le ho hlahloba li-RNA tse chitja, sehlopha sa limolek'hule tsa RNA tse etsang li-loops tse koetsoeng ka lebaka la liketsahalo tse sa lumellaneng le li-canonical, ho fana ka RNA ena ka botsitso bo eketsehileng. Le ha li-circRNA tse ling li bonts'itsoe hore li sebetsa joalo ka liponche tsa microRNA, li ts'oara li-microRNA le ho li thibela ho laola li-mRNA tsa tsona, li-circRNA tse ling li ka sebelisana le liprotheine, tsa fetola liphatsa tsa lefutso, kapa tsa ba le likarolo lits'ebetsong tsa cellular. Tlhahlobo ea polelo ea circRNA e fana ka leseli mabapi le likarolo tsa taolo ea limolek'hule tsena le bohlokoa ba tsona lits'ebetsong tse fapaneng tsa lisele, methati ea nts'etsopele, le maemo a mafu, e tlatsetsang kutloisisong e tebileng ea ho rarahana ha taolo ea RNA maemong a polelo ea lefutso.
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Tatelano e Feletseng ea Transcriptome - Illumina
Tatelano e felletseng ea transcriptome e fana ka mokhoa o felletseng oa ho tsebahatsa limolek'hule tse fapaneng tsa RNA, tse kenyelletsang likhoutu (mRNA) le li-RNA tse sa khoutang (lncRNA, circRNA, le miRNA). Mokhoa ona o hapa transcriptome eohle ea lisele tse itseng ka nako e itseng, e lumellang kutloisiso e akaretsang ea lits'ebetso tsa cellular. E boetse e tsejoa e le "total RNA sequencing," e ikemiselitse ho senola marang-rang a rarahaneng a taolo boemong ba transcriptome, ho nolofalletsa tlhahlobo e tebileng joalo ka tlholisano ea endogenous RNA (ceRNA) le tlhahlobo e kopaneng ea RNA. Sena se tšoaea mohato oa pele o lebisang ho sebopeho se sebetsang, haholo-holo ho manolleng marang-rang a taolo a kenyelletsang litšebelisano tsa ceRNA tse thehiloeng ho circRNA-miRNA-mRNA.
