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Single-nuclei RNA Sequencing
Kuvandudzwa kwekutorwa kwesero rimwe chete uye tsika dzekuvaka raibhurari, pamwe chete nepamusoro-soro kutevedzana, yakashandura zvidzidzo zvejini padanho resero. Kubudirira uku kunobvumira kuongororwa kwakadzama uye kwakadzama kwehuwandu hwemasero akaomarara, kukunda zvipingamupinyi zvine chekuita neavhareji yekutaura kwemajini pamusoro pemasero ese uye kuchengetedza heterogeneity yechokwadi mukati mehuwandu hwevanhu. Nepo single-cell RNA sequencing (scRNA-seq) ine mabhenefiti asingarambike, inosangana nematambudziko mune mamwe matissue uko kugadzirwa kwesero-sero kumiswa kunoratidza kwakaoma uye kunoda sampuli nyowani. PaBMKGene, tinogadzirisa chipingamupinyi ichi nekupa imwechete-nucleus RNA sequencing (snRNA-seq) tichishandisa iyo-ye-iyo-ye-iyo 10X Genomics Chromium tekinoroji. Iyi nzira inowedzera kuwanda kwemasamples anogoneka kuongororo yezvinyorwa padanho resero rimwe chete.
Kuparadzaniswa kweiyo nuclei kunoitwa kuburikidza neyakagadziridzwa 10X Genomics Chromium chip, ine sere-chiteshi microfluidics system ine kaviri kuyambuka. Mukati megadziriro iyi, mabheji ejeri anosanganisira mabhakodhi, maprimers, enzymes, uye nucleus imwe chete yakavharidzirwa mumadonhwe emafuta e-nanoliter, achigadzira Gel Bead-in-Emulsion (GEM). Kutevera kuumbwa kweGEM, cell lysis uye barcode kuburitswa kunoitika mukati meGEM yega yega. Zvadaro, mRNA mamorekuru anonyorerwa reverse mucDNAs, inobatanidza 10X barcode uye Unique Molecular Identifiers (UMIs). Aya ma cDNAs anobva aiswa pasi peyakajairwa kuteedzera raibhurari kuvaka, kufambisa kwakasimba uye kwakadzama kuongororwa kwemajini ekutaura profiles padanho resero rimwe.
Platform: 10 × Genomics Chromium uye Illumina NovaSeq Platform
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10x Genomics Visium Spatial Transcriptome
Spatial transcriptomics tekinoroji yekucheka-kumucheto iyo inobvumira vaongorori kuti vaongorore maitiro ekutaura kwemajini mukati mematishu vachichengetedza mamiriro avo epamhepo. Imwe puratifomu ine simba mune ino domain ndeye 10x Genomics Visium yakasanganiswa neIllumina kutevedzana. Iyo musimboti we10X Visium iri pane yakasarudzika chip ine yakasarudzika nzvimbo yekubata inoiswa zvikamu zvetishu. Iyi nzvimbo yekubata ine barcoded mavara, imwe neimwe inoenderana nenzvimbo yakasarudzika mukati mematishu. Iwo akabatwa maRNA mamorekuru kubva munyama anobva anyorwa neakasarudzika mamorekuru identifiers (UMIs) panguva yekudzokesa kunyorwa maitiro. Aya mabarcoded spots uye UMIs anogonesa chaiyo spatial mepu uye quantification yegeneti kutaura pane imwechete-sero resolution. Iko kusanganiswa kwe spatially barcoded samples uye UMIs inovimbisa kurongeka uye kujeka kweiyo data inogadzirwa. Nekushandisa iyi Spatial Transcriptomics tekinoroji, vaongorori vanogona kuwana nzwisiso yakadzama yekurongeka kwenzvimbo yemasero uye kupindirana kwakaoma kwema molecular kunoitika mukati mematishu, zvichipa ruzivo rwakakosha mumatanho ari pasi pemaitiro ezvipenyu mundima dzakawanda, kusanganisira oncology, neuroscience, developmental biology, immunology. , uye zvidzidzo zvebotanical.
Platform: 10X Genomics Visium uye Illumina NovaSeq
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Yakazara-Kureba mRNA Sequencing-Nanopore
Nepo NGS-yakavakirwa mRNA sequencing iri chishandiso chinogoneka chekuenzanisa gene kutaura, kuvimba kwayo nekuverenga kupfupi kunodzora kushanda kwayo mukuongorora kwakaoma kwezvinyorwa. Kune rimwe divi, nanopore sequencing inoshandisa tekinoroji yekuverenga kwenguva refu, ichigonesa kutevedzana kwehurefu hwakazara mRNA zvinyorwa. Iyi nzira inogonesa kuongororwa kwakadzama kweimwe nzira splicing, gene fusions, poly-adenylation, uye quantification yemRNA isoforms.
Nanopore sequencing, nzira inotsamira pane nanopore single-morekuru real-time magetsi masaini, inopa mhedzisiro munguva chaiyo-nguva. Inotungamirwa nemapuroteni emota, DNA ine tambo mbiri inosunga kune nanopore mapuroteni akaiswa mubiofilm, achisununguka sezvo ichipfuura nemu nanopore chiteshi pasi pemutsauko wevoltage. Iwo akasiyana masaini emagetsi anogadzirwa nemabhesi akasiyana paDNA strand anoonekwa uye akaiswa muchikamu-chaiyo-nguva, kufambisa kwakaringana uye kuenderera nucleotide sequencing. Iyi nzira yekuvandudza inokunda zvipimo zvekuverenga kwenguva pfupi uye inopa chikuva chine simba chekuomesesa genomic kuongororwa, kusanganisira yakaoma transcriptomic zvidzidzo, zvine mhedzisiro yazvino.
Platform: Nanopore PromethION 48
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Yakazara-kureba mRNA kutevedzana -PacBio
Nepo NGS-yakavakirwa mRNA sequencing iri chishandiso chinogoneka chekuenzanisa gene kutaura, kuvimba kwayo nekuverenga kupfupi kunodzora kushandiswa kwayo mukuomarara kwekuongorora. Kune rimwe divi, PacBio sequencing (Iso-Seq) inoshandisa tekinoroji yekuverenga kwenguva refu, ichigonesa kutevedzana kwehurefu hwakazara mRNA zvinyorwa. Iyi nzira inogonesa kuongororwa kwakadzama kweimwe nzira splicing, gene fusions, uye poly-adenylation. Nekudaro, kune dzimwe sarudzo dzegene expression quantification nekuda kwehuwandu hwedata hunodiwa. PacBio sequencing tekinoroji inotsamira pane imwe-morekuru, chaiyo-nguva (SMRT) kutevedzana, ichipa mukana wakasiyana mukutora yakazara-kureba mRNA zvinyorwa. Iyi nzira yekuvandudza inosanganisira kushandisa zero-mode waveguides (ZMWs) uye matsime ane microfabricated anogonesa kutarisisa kwechokwadi-nguva yeDNA polymerase chiitiko panguva yekutevedzana. Mukati meaya maZMWs, PacBio's DNA polymerase inogadzira tambo inopindirana yeDNA, ichigadzira kuverenga kwakareba kunosvika kuzere kwezvinyorwa zvemRNA. PacBio oparesheni muCircular Consensus sequencing (CCS) modhi inosimudzira kurongeka nekudzokorodza kuteedzera mamorekuru mamwe chete. Iwo akagadzirwa HiFi anoverenga ane huchokwadi hunofananidzwa neNGS, ichienderera mberi ichipa kune yakazara uye yakavimbika ongororo yeakaoma transcriptomic maficha.
Platform: PacBio Sequel II; PacBio Revio
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Eukaryotic mRNA Sequencing-NGS
mRNA sequencing, tekinoroji inosiyana-siyana, inopa simba rakazara kunyorwa kweese mRNA zvinyorwa mukati memasero pasi pemamiriro chaiwo. Nemashandisirwo ayo akasiyana-siyana, ichi chekucheka-kumucheto chishandiso chinovhenekera chakaomesesa gene kutaura profiles, majini zvimiro, uye mamorekuru maitiro ane chekuita neakasiyana biological process. Yakagamuchirwa zvakanyanya mukutsvagisa kwakakosha, kuongororwa kwekiriniki, uye kukura kwezvinodhaka, kutevedzana kwemRNA kunopa nzwisiso mukuomesesa kweserura dynamics uye genetic regulation, ichimutsa kuda kuziva nezve kugona kwayo muzvikamu zvakasiyana.
Platform: Illumina NovaSeq X; DNBSEQ-T7
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Non-Reference based mRNA Sequencing-NGS
mRNA sequencing inogonesa kukwenenzverwa kwezvese mRNA zvinyorwa mukati memasero pasi pemamiriro chaiwo. Iyi tekinoroji yekucheka-kumucheto inoshanda sechishandiso chine simba, kuvheneka akaomesesa gene kutaura profiles, majini zvimiro, uye mamorekuru maitiro ane chekuita neakasiyana maitiro ebhayoloji. Yakagamuchirwa zvakanyanya mukutsvagisa kwakakosha, kuongororwa kwekiriniki, uye kukura kwezvinodhaka, kutevedzana kwemRNA kunopa manzwisisiro mukati mekuoma kwemaserura dynamics uye genetic regulation.
Platform: Illumina NovaSeq X; DNBSEQ-T7
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Yakareba Non-coding Sequencing-Illumina
Marefu asiri ekukodha maRNA (lncRNAs) akareba kupfuura mazana maviri nucleotides ane mashoma ekukodha anogona uye ari akakosha zvinhu mukati isiri-coding RNA. Inowanikwa mune nucleus uye cytoplasm, aya maRNA anoita basa rakakosha mune epigenetic, transcriptal, uye post-transcriptal regulation, zvichisimbisa kukosha kwavo mukugadzira maserura uye mamorekuru maitiro. LncRNA sequencing chishandiso chine simba mukusiyanisa Cell, Ontogenesis, uye Zvirwere zvevanhu.
Platform: Illumina NovaSeq
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Diki RNA Sequencing-Illumina
Madiki eRNA (sRNA) mamorekuru, anosanganisira microRNAs (miRNAs), maRNA madiki anopindira (siRNAs), uye piwi-inopindirana RNAs (piRNAs). Pakati peaya, miRNAs, yakatenderedza 18-25 nucleotides kureba, inonyanya kukosha pamabasa avo akakosha ekutonga mumaserura akasiyana maitiro. Nematishu-chaiwo uye nhanho-chaiwo maitiro ekutaura, miRNAs inoratidza kuchengetedza kwepamusoro pamarudzi akasiyana.
Platform: Illumina NovaSeq
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CircRNA Sequencing-Illumina
Circular RNA sequencing (circRNA-seq) ndeyekufananidza uye kuongorora maRNA akatenderera, kirasi yemamorekuru eRNA anoumba zvishwe zvakavharwa nekuda kwezviitiko zvisiri zvecanonical splicing, zvichipa RNA iyi nekuwedzera kugadzikana. Nepo mamwe ma circRNA akaratidzwa kuita semicroRNA sponges, sequering microRNAs uye kuvadzivirira kubva pakudzora vavanoda mRNAs, mamwe ma circRNA anogona kudyidzana nemapuroteni, modulate gene expression, kana kuita mabasa mumaserura. circRNA kutaura kwekuongorora kunopa ruzivo mumabasa ekutonga emamorekuru aya uye kukosha kwawo mumaitiro akasiyana-siyana emasero, nhanho dzekusimudzira, uye mamiriro echirwere, zvichipa kunzwisisa kwakadzama kwekuoma kwemutemo weRNA mumamiriro ekutaura kwejeni.
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Yese Transcriptome Sequencing - Illumina
Yese transcriptome sequencing inopa yakazara nzira yekunyora akasiyana mamorekuru eRNA, anosanganisira coding (mRNA) uye isiri-coding RNAs (lncRNA, circRNA, uye miRNA). Iyi tekinoroji inobata iyo yese transcriptome yemamwe maseru panguva yakapihwa, ichibvumira kunzwisisa kwakazara kwemaserura maitiro. Iyo inozivikanwawo se "yakazara RNA kutevedzana," ine chinangwa chekufumura akaomesesa ekutonga network padanho rekunyora, ichigonesa kuongororwa kwakadzama sekukwikwidza endo native RNA (ceRNA) uye yemubatanidzwa RNA kuongororwa. Izvi zvinoratidzira danho rekutanga rekuita basa rekuita, kunyanya mukufumura mataneti ekudzora anosanganisira circRNA-miRNA-mRNA-yakavakirwa ceRNA kusangana.
