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Kev Txheeb Xyuas RNA Ib Leeg
Kev tsim cov txheej txheem ntawm kev ntes ib lub cell thiab kev tsim cov tsev qiv ntawv, ua ke nrog kev ua kom muaj txiaj ntsig zoo, tau hloov pauv txoj kev tshawb fawb txog kev qhia txog noob caj noob ces ntawm theem cell. Qhov kev tshawb pom no tso cai rau kev tshuaj xyuas tob dua thiab ntau dua ntawm cov pej xeem cell nyuaj, kov yeej cov kev txwv uas cuam tshuam nrog kev nruab nrab ntawm kev qhia txog noob caj noob ces hla txhua lub cell thiab khaws cia qhov sib txawv tiag tiag hauv cov pej xeem no. Thaum kev ua kom muaj noob caj noob ces ib lub cell (scRNA-seq) muaj qhov zoo tsis lees paub, nws ntsib cov teeb meem hauv qee cov nqaij uas kev tsim cov noob caj noob ces ib lub cell nyuaj thiab xav tau cov qauv tshiab. Ntawm BMKGene, peb daws qhov teeb meem no los ntawm kev muab cov noob caj noob ces ib lub nucleus RNA (snRNA-seq) siv cov thev naus laus zis 10X Genomics Chromium uas siv niaj hnub no. Txoj hauv kev no ua rau cov qauv ntawm cov qauv dav dua rau kev tshuaj xyuas transcriptome ntawm theem cell ib lub cell.
Kev rho tawm cov nuclei tau ua tiav los ntawm lub 10X Genomics Chromium chip tshiab, uas muaj yim-channel microfluidics system nrog ob chav hla. Hauv lub system no, cov hlaws gel uas muaj cov barcodes, primers, enzymes, thiab ib lub nucleus raug encapsulated hauv nanoliter-sized roj tee, tsim Gel Bead-in-Emulsion (GEM). Tom qab GEM tsim, cell lysis thiab barcode tso tawm tshwm sim hauv txhua GEM. Tom qab ntawd, mRNA molecules dhau los ua reverse transcription rau hauv cDNAs, suav nrog 10X barcodes thiab Unique Molecular Identifiers (UMIs). Cov cDNAs no tom qab ntawd raug rau kev tsim cov tsev qiv ntawv sequencing txheem, pab txhawb kev tshawb nrhiav zoo thiab kev tshawb nrhiav ntawm cov qauv qhia txog gene ntawm ib lub cell.
Platform: 10 × Genomics Chromium thiab Illumina NovaSeq Platform
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Kev Tshawb Fawb Txog MRNA Tag Nrho-Nanopore
Txawm hais tias NGS-based mRNA sequencing yog ib qho cuab yeej siv tau ntau yam rau kev ntsuas qhov kev qhia tawm ntawm cov noob caj noob ces, nws txoj kev vam khom rau kev nyeem luv luv txwv nws txoj kev ua haujlwm hauv kev tshuaj xyuas cov ntawv sau ua ke. Ntawm qhov tod tes, nanopore sequencing siv cov thev naus laus zis nyeem ntev, ua rau muaj kev txheeb xyuas cov ntawv sau mRNA tag nrho. Txoj hauv kev no pab txhawb kev tshawb nrhiav txog lwm txoj hauv kev splicing, gene fusions, poly-adenylation, thiab kev ntsuas ntawm mRNA isoforms.
Kev ntsuas Nanopore sequencing, ib txoj kev uas siv cov teeb liab hluav taws xob nanopore ib lub molecule tiag tiag, muab cov txiaj ntsig tiag tiag. Kev coj los ntawm cov protein motor, ob txoj hlua DNA khi rau cov protein nanopore uas muab tso rau hauv biofilm, unwinding thaum nws hla dhau lub nanopore channel nyob rau hauv qhov sib txawv voltage. Cov teeb liab hluav taws xob sib txawv uas tsim los ntawm cov hauv paus sib txawv ntawm DNA strand raug kuaj pom thiab faib ua pawg tiag tiag, pab txhawb kev ntsuas nucleotide kom raug thiab txuas ntxiv mus. Txoj hauv kev tshiab no kov yeej cov kev txwv luv luv thiab muab lub platform dynamic rau kev tshuaj xyuas genomic nyuaj, suav nrog kev tshawb fawb transcriptomic nyuaj, nrog cov txiaj ntsig tam sim ntawd.
Platform: Nanopore PromethION 48
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Kev txheeb xyuas mRNA tag nrho -PacBio
Txawm hais tias NGS-based mRNA sequencing yog ib qho cuab yeej siv tau ntau yam rau kev ntsuas qhov kev qhia tawm ntawm cov noob caj noob ces, nws qhov kev vam khom rau kev nyeem luv luv txwv tsis pub siv nws hauv kev tshuaj xyuas cov ntawv sau ua ke. Ntawm qhov tod tes, PacBio sequencing (Iso-Seq) siv cov thev naus laus zis nyeem ntev, ua rau muaj kev txheeb xyuas cov ntawv sau mRNA tag nrho. Txoj hauv kev no ua rau muaj kev tshawb nrhiav dav dav ntawm lwm txoj hauv kev splicing, gene fusions, thiab poly-adenylation. Txawm li cas los xij, muaj lwm txoj hauv kev xaiv rau kev ntsuas qhov kev qhia tawm ntawm cov noob caj noob ces vim muaj cov ntaub ntawv ntau heev. PacBio sequencing thev naus laus zis vam khom ib leeg-molecule, real-time (SMRT) sequencing, muab qhov zoo sib xws hauv kev ntes cov ntawv sau mRNA tag nrho. Txoj hauv kev tshiab no suav nrog kev siv xoom-mode waveguides (ZMWs) thiab microfabricated wells uas ua rau muaj kev soj ntsuam tiag tiag ntawm DNA polymerase kev ua haujlwm thaum lub sijhawm txheeb xyuas. Hauv cov ZMWs no, PacBio's DNA polymerase synthesizes ib txoj hlua DNA sib txuas, tsim cov ntawv nyeem ntev uas hla tag nrho cov ntawv sau mRNA. Kev ua haujlwm ntawm PacBio hauv hom Circular Consensus sequencing (CCS) ua rau muaj qhov tseeb ntxiv los ntawm kev rov ua dua qhov kev txheeb xyuas tib lub molecule. Cov ntawv nyeem HiFi uas tsim tawm muaj qhov tseeb sib piv rau NGS, ntxiv rau kev pab txhawb rau kev tshuaj xyuas kom meej thiab txhim khu kev qha ntawm cov yam ntxwv transcriptomic nyuaj.
Platform: PacBio Sequel II; PacBio Revio: koj puas xav tau ntau tus thwjtim?
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Eukaryotic mRNA Sequencing-NGS
mRNA sequencing yog ib txoj kev siv cov txheej txheem tom ntej los qhia qhov muaj thiab qhov ntau ntawm RNA molecules hauv cov qauv tshuaj lom neeg, muab ib daim duab ntawm kev qhia tawm gene hauv cov qauv.
Nrog nws cov ntawv thov dav dav, cov cuab yeej txiav tshiab no qhia txog cov qauv qhia txog cov noob caj noob ces, cov qauv noob caj noob ces, thiab cov txheej txheem molecular uas cuam tshuam nrog ntau yam txheej txheem ntawm lub cev.
Cov txheej txheem no tau siv dav hauv kev tshawb fawb, kev kuaj mob hauv tsev kho mob, thiab kev tsim tshuaj, vim nws muab kev nkag siab txog qhov nyuaj ntawm cov cellular dynamics thiab kev tswj hwm caj ces, muab lub hauv paus ruaj khov los ua kom muaj kev xav paub txog nws lub peev xwm ntawm ntau qhov chaw sib txawv.
Cov platforms muaj: Illumina NovaSeq X; DNBSEQ-T7
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Kev Siv mRNA Sequencing Uas Tsis Yog Siv Los Xam-NGS
Kev txheeb xyuas mRNA ua rau muaj kev nkag siab txog txhua cov ntawv sau mRNA hauv cov cell raws li cov xwm txheej tshwj xeeb. Cov thev naus laus zis tshiab no ua haujlwm ua ib qho cuab yeej muaj zog, qhia txog cov qauv qhia txog kev qhia txog cov noob caj noob ces, cov qauv noob caj noob ces, thiab cov txheej txheem molecular uas cuam tshuam nrog ntau yam txheej txheem ntawm lub cev. Siv dav hauv kev tshawb fawb tseem ceeb, kev kuaj mob hauv tsev kho mob, thiab kev tsim tshuaj, mRNA sequencing muab kev nkag siab txog qhov nyuaj ntawm cov cellular dynamics thiab kev tswj hwm noob caj noob ces.
Platform: Illumina NovaSeq X; DNBSEQ-T7
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Kev Ua Sequencing Ntev Uas Tsis Yog Coding-Illumina
Cov RNA ntev uas tsis yog coding (lncRNAs) yog cov ntev dua 200 nucleotides uas muaj qhov tsawg kawg nkaus coding potential thiab yog cov ntsiab lus tseem ceeb hauv cov RNA uas tsis yog coding. Pom nyob rau hauv lub nucleus thiab cytoplasm, cov RNAs no ua lub luag haujlwm tseem ceeb hauv epigenetic, transcriptional, thiab post-transcriptional regulation, qhia txog lawv qhov tseem ceeb hauv kev tsim cov txheej txheem cellular thiab molecular. LncRNA sequencing yog ib qho cuab yeej muaj zog hauv Cell differentiation, Ontogenesis, thiab Human kab mob.
Platform: Illumina NovaSeq X
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Kev Tshawb Fawb Me Me RNA-Illumina
RNA me me yog ib pawg ntawm cov RNA molecule uas suav nrog microRNAs (miRNAs), RNAs cuam tshuam me me (siRNAs), thiab piwi-interacting RNAs (piRNAs). Ntawm cov no, miRNAs, kwv yees li 18-25 nucleotides ntev, yog qhov tseem ceeb rau lawv lub luag haujlwm tseem ceeb hauv ntau yam txheej txheem cellular. Nrog rau cov qauv qhia tawm tshwj xeeb rau cov ntaub so ntswg thiab theem, miRNAs qhia txog kev txuag siab thoob plaws ntau hom tsiaj sib txawv.
Platform: Illumina NovaSeq
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Kev Txheeb Xyuas CircRNA-Illumina
Kev txheeb xyuas RNA voj voog (circRNA-seq) profiles thiab tshuaj xyuas cov RNA voj voog, ib chav kawm ntawm RNA molecules uas tsim cov voj voog kaw vim yog cov xwm txheej splicing tsis yog canonical, muab cov RNA no nrog kev ruaj khov ntxiv. Txawm hais tias qee qhov circRNAs tau pom tias ua haujlwm ua microRNA sponges, cais microRNAs thiab tiv thaiv lawv los ntawm kev tswj hwm lawv lub hom phiaj mRNAs, lwm cov circRNAs yuav cuam tshuam nrog cov protein, hloov kho kev qhia gene, lossis muaj lub luag haujlwm hauv cov txheej txheem cellular. Kev tshuaj xyuas kev qhia circRNA muab kev nkag siab rau hauv lub luag haujlwm tswj hwm ntawm cov molecules no thiab lawv qhov tseem ceeb hauv ntau yam txheej txheem cellular, theem kev loj hlob, thiab cov mob, pab txhawb rau kev nkag siab tob dua ntawm qhov nyuaj ntawm kev tswj hwm RNA hauv cov ntsiab lus ntawm kev qhia gene.
Platform: Illumina Novaseq X
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Kev Txheeb Xyuas Tag Nrho Cov Ntawv Sau - Illumina
Wole transcriptome sequencing muab ib txoj hauv kev dav dav rau kev txheeb xyuas ntau yam RNA molecules, suav nrog coding (mRNA) thiab non-coding RNAs (lncRNA, circRNA, thiab miRNA). Cov txheej txheem no ntes tag nrho cov transcriptome ntawm cov cell tshwj xeeb rau lub sijhawm, uas tso cai rau kev nkag siab txog cov txheej txheem cellular.
Cov platforms muaj: Illumina Novaseq X
