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Single- nuclei RNA Ulandelelwano
Uphuhliso lokubanjwa kweseli enye kunye nobuchule bokwakha ithala leencwadi, ludityaniswa nolandelelwano oluphezulu, luguqule izifundo zokuchazwa kofuzo kwinqanaba leseli. Le mpumelelo ivumela uhlalutyo olunzulu nolubanzi ngakumbi lweeseli ezintsonkothileyo, ukoyisa imida eyayanyaniswa ne-avareji yembonakalo yemfuza kuzo zonke iiseli kunye nokugcina iheterogeneity yokwenyani phakathi kwaba bantu. Ngelixa ulandelelwano lwe-RNA yeseli enye (scRNA-seq) ineengenelo ezingenakuphikiswa, ihlangabezana nemiceli mngeni kwizicubu ezithile apho ukudalwa kokumiswa kweseli enye kubonakala kunzima kwaye kufuna iisampuli ezintsha. Kwi-BMKGene, sijongana nalo mqobo ngokunikezela ngolandelelwano lwe-nucleus ye-RNA enye (snRNA-seq) sisebenzisa itekhnoloji ye-10X Genomics Chromium yanamhlanje. Le ndlela yokwandisa i-spectrum yeesampuli ezivumelekileyo kuhlalutyo lwe-transcriptome kwinqanaba leseli enye.
Ukwahlukaniswa kwe-nuclei kufezekiswa nge-10X ye-Genomics ye-Chromium chip entsha, equkethe inkqubo ye-microfluidics yamajelo asibhozo kunye nokuwela kabini. Ngaphakathi kwesi sixokelelwano, ubuhlalu be-gel obubandakanya i-barcodes, i-primers, i-enzymes, kunye ne-nucleus enye ifakwe kwi-nanoliter-sized oil drops, eyenza i-Gel Bead-in-Emulsion (GEM). Ukulandela ukubunjwa kwe-GEM, i-cell lysis kunye nokukhululwa kwebhakhowudi kwenzeka ngaphakathi kwe-GEM nganye. Emva koko, iimolekyuli ze-mRNA ziphinda zikhutshelwe umva kwii-cDNAs, ezibandakanya i-10X yeebhakhowudi kunye ne-Unique Molecular Identifiers (UMIs). Ezi cDNAs ke ngoko ziphantsi kolwakhiwo lwethala leencwadi lolandelelwano olusemgangathweni, luququzelela ukuphononongwa okuqinileyo nolubanzi lweeprofayili zokuchazwa kofuzo kwinqanaba leseli enye.
Iqonga: 10× Genomics Chromium kunye Illumina NovaSeq Platform
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I-10x ye-Genomics yeVisium ye-Spatial Transcriptome
I-Spatial transcriptomics yitekhnoloji enqamlezileyo evumela abaphandi ukuba baphande iipatheni zokubonakaliswa kofuzo ngaphakathi kwezicubu ngelixa begcina imeko yabo yendawo. Iqonga elinye elinamandla kule domain yi-10x Genomics Visium edityaniswa nokulandelelana kwe-Illumina. Umgaqo we-10X Visium ulele kwi-chip ekhethekileyo enendawo ekhethiweyo yokubamba apho amacandelo e-tissue abekwe khona. Le ndawo yokubamba iqulethe iindawo ezinebhakhowudi, nganye ihambelana nendawo eyodwa ngaphakathi kwethishu. Iimolekyuli ze-RNA ezibanjiweyo kwiithishu zibhalwa emva koko zibhalwe nge-molecular identifiers (UMIs) ezikhethekileyo ngexesha lenkqubo yokubhala umva. Ezi ndawo zinebhakhowudi kunye nee-UMIs zenza imephu yendawo echanekileyo kunye nobungakanani bokubonakaliswa kofuzo kwisisombululo seseli enye. Ukudityaniswa kweesampulu ezinebhakhowudi yendawo kunye nee-UMIs ziqinisekisa ukuchaneka kunye nokuchaneka kwedatha eyenziweyo. Ngokusebenzisa le teknoloji ye-Spatial Transcriptomics, abaphandi banokufumana ukuqonda okunzulu malunga nentlangano yendawo yeeseli kunye nentsebenziswano eyinkimbinkimbi ye-molecular eyenzeka ngaphakathi kwezicubu, inika ulwazi oluxabisekileyo kwiindlela eziphantsi kweenkqubo zebhayoloji kwiinkalo ezininzi, kuquka i-oncology, neuroscience, i-biology yophuhliso, i-immunology. , kunye nezifundo zebhotani.
Iqonga: I-10X Genomics Visium kunye ne-Illumina NovaSeq
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Ubude obupheleleyo be-mRNA Ukulandelelana-Nanopore
Ngelixa ulandelelwano olusekwe kwi-NGS ye-mRNA sisixhobo esiguquguqukayo sokulinganisa ukubonakaliswa kofuzo, ukuxhomekeka kwayo ekufundeni okufutshane kuthintela ukusebenza kwayo kuhlalutyo oluntsonkothileyo lwe-transcriptomic. Kwelinye icala, ulandelelwano lwe-nanopore lusebenzisa itekhnoloji yokufunda ixesha elide, ivumela ulandelelwano loshicilelo olupheleleyo lwe-mRNA. Le ndlela iququzelela ukuphononongwa okubanzi kwezinye iindlela zokudityaniswa, ukudityaniswa kwemfuza, i-poly-adenylation, kunye nobungakanani be-mRNA isoforms.
Ukulandelelana kwe-Nanopore, indlela exhomekeke kwi-nanopore-molecule ye-real-time real-time signal signals, inika iziphumo ngexesha langempela. Ukukhokelwa ziiprotheyini zeemoto, i-DNA ephindwe kabini ibophelela kwiiprotheni ze-nanopore ezifakwe kwi-biofilm, ihlanjululwe njengoko idlula kumjelo we-nanopore phantsi kokwahlukana kombane. Izibonakaliso zombane ezihlukeneyo eziveliswa ziziseko ezahlukeneyo kwi-DNA strand zichongwa kwaye zihlelwe ngexesha langempela, ziququzelele ukulandelelana kwe-nucleotide echanekileyo kunye nokuqhubekayo. Le ndlela yokuvelisa izinto ezintsha iyoyisa imida yokufunda okufutshane kwaye ibonelela ngeqonga eliguqukayo lohlalutyo oluntsonkothileyo lwe-genomic, kubandakanywa nezifundo ezintsonkothileyo ze-transcriptomic, ezineziphumo ezikhawulezayo.
Iqonga: iNanopore PromethION 48
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Ulandelelwano olupheleleyo lwe-mRNA -PacBio
Ngelixa ulandelelwano olusekwe kwi-NGS ye-mRNA sisixhobo esiguquguqukayo sokulinganisa ukubonakaliswa kofuzo, ukuthembela kufundo olufutshane kuthintela ukusetyenziswa kwayo kuhlalutyo oluntsonkothileyo lwe-transcriptomic. Ngakolunye uhlangothi, i-PacBio sequencing (Iso-Seq) isebenzisa iteknoloji yokufunda ixesha elide, eyenza ukulandelelana kwemibhalo egcweleyo ye-mRNA. Le ndlela iququzelela ukuphononongwa okubanzi kwezinye iindlela zokudityaniswa, ukudityaniswa kwemfuza, kunye ne-poly-adenylation. Nangona kunjalo, kukho ezinye iinketho zokuchazwa kohlobo lobungakanani ngenxa yedatha ephezulu efunekayo. Itekhnoloji yokulandelelanisa i-PacBio ixhomekeke kwi-molecule enye, i-real-time (SMRT) yokulandelelana, inika inzuzo eyahlukileyo ekubanjeni imibhalo ye-mRNA yobude obupheleleyo. Le ndlela entsha ibandakanya ukusebenzisa i-zero-mode waveguides (ZMWs) kunye nemingxuma emincinci eyenza ukubonwa kwexesha langempela lomsebenzi we-DNA polymerase ngexesha lokulandelelana. Ngaphakathi kwezi ZMWs, iPacBio's DNA polymerase idibanisa umsonto ohambelanayo weDNA, ivelisa ufundo olude olufikelela kuyo yonke imibhalo ye-mRNA. Ukusebenza kwe-PacBio kwimodi yeSetyhula ye-Consensus sequencing (CCS) iphucula ukuchaneka ngokulandelelana ngokuphindaphindiweyo i-molecule efanayo. Ukufundwa kwe-HiFi eveliswayo kunokuchaneka okuthelekiswa ne-NGS, igalelo elingakumbi kuhlalutyo olubanzi noluthembekileyo lweempawu ezintsonkothileyo ze-transcriptomic.
Iqonga: I-PacBio Sequel II; I-PacBio Revio
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I-Eukaryotic mRNA Sequencing-NGS
Ulandelelwano lwe-mRNA, itekhnoloji eguquguqukayo, ixhobisa iprofayili ebanzi yazo zonke iikopi ze-mRNA ngaphakathi kweeseli phantsi kweemeko ezithile. Ngokusetyenziswa kwaso kuluhlu olubanzi, esi sixhobo sokusika sityhila iiprofayili ezintsonkothileyo zokuchazwa kofuzo, ulwakhiwo lwemfuza, kunye neendlela zemolekyuli ezinxulumene neenkqubo ezahlukeneyo zebhayoloji. Yamkelwa ngokubanzi kuphando olusisiseko, uxilongo lweklinikhi, kunye nophuhliso lwamachiza, ulandelelwano lwe-mRNA lubonelela ngokuqonda kubuntsokotha bamandla eselula kunye nolawulo lwemfuzo, ivuselela umdla malunga nokubanakho kwayo kwiinkalo ezahlukeneyo.
Iqonga: Illumina NovaSeq X; I-DNBSEQ-T7
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I-Non-Reference based mRNA Sequencing-NGS
Ulandelelwano lwe-mRNA luxhobisa iprofayili ebanzi yazo zonke iikopi ze-mRNA ngaphakathi kweeseli phantsi kweemeko ezithile. Le teknoloji ye-cutting-edge isebenza njengesixhobo esinamandla, ityhila iiprofayili zegenesis ezintsonkothileyo, ulwakhiwo lwemfuza, kunye neendlela zemolekyuli ezinxulumene neenkqubo ezahlukeneyo zebhayoloji. Yamkelwa ngokubanzi kuphando olusisiseko, uxilongo lweklinikhi, kunye nophuhliso lwamachiza, ulandelelwano lwe-mRNA lubonelela ngokuqonda kubuntsonkotha bokutshintshatshintsha kweeseli kunye nolawulo lwemfuzo.
Iqonga: Illumina NovaSeq X; I-DNBSEQ-T7
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Ixesha elide Non-coding Ulandelelwano-Illumina
Ii-RNA ezinde ezingabhaliyo iikhowudi (lncRNAs) zinde kune-200 nucleotides ezinesakhono sokwenza iikhowudi ezincinci kwaye zizinto ezibalulekileyo ngaphakathi kwe-RNA engakhowudi. Ifunyanwe kwi-nucleus kunye ne-cytoplasm, ezi RNAs zidlala indima ebalulekileyo kwi-epigenetic, transcriptal, kunye ne-post-transcriptal regulation, igxininisa ukubaluleka kwazo ekuqulunqeni iinkqubo zeselula kunye ne-molecular. Ukulandelelana kwe-LncRNA sisixhobo esinamandla kwi-Cell differentiation, i-Ontogenesis, kunye nezifo zabantu.
Iqonga: Illumina NovaSeq
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I-RNA encinci Ukulandelelana-Illumina
Iimolekyuli ezincinci ze-RNA (sRNA), ziquka i-microRNAs (miRNAs), i-RNAs encinci ephazamisayo (i-siRNAs), kunye ne-piwi-interacting RNAs (piRNAs). Phakathi kwezi, ii-miRNAs, malunga ne-18-25 nucleotides ubude, ziphawuleka ngakumbi kwiindima zazo zolawulo olubalulekileyo kwiinkqubo ezahlukeneyo zeselula. Ngeepateni zokuchaza ezithe ngqo kunye nenqanaba elithile, ii-miRNAs zibonisa ulondolozo oluphezulu kuzo zonke iintlobo ezahlukeneyo.
Iqonga: Illumina NovaSeq
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CircRNA Ulandelelwano-Illumina
Ulandelelwano lwe-RNA yesetyhula (i-circRNA-seq) kukuchaza iphrofayili kunye nokuhlalutya i-RNAs esetyhula, iklasi yee-molecule ze-RNA ezenza i-loops evaliweyo ngenxa yeziganeko ze-non-canonical splicing, ukubonelela le RNA ngokunyuka kozinzo. Ngelixa ezinye ii-circRNAs zibonakaliswe ukuba zisebenza njengeziponji ze-microRNA, zihlutha ii-microRNAs kwaye zibathintele ekulawuleni i-mRNAs ekujoliswe kuyo, ezinye ii-circRNAs zinokusebenzisana neeprotheni, zimodareyitha imbonakalo yemfuza, okanye zibe neendima kwiinkqubo zeselula. Uhlalutyo lwentetho ye-circRNA lubonelela ngengqiqo kwiindima zokulawula ezi molekyuli kunye nokubaluleka kwazo kwiinkqubo ezahlukeneyo zeselula, amanqanaba ophuhliso, kunye neemeko zezifo, ezinegalelo ekuqondeni okunzulu kobunzima bolawulo lwe-RNA kumxholo we-gene expression.
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Ulandelelwano olupheleleyo lweTranscriptome-Illumina
Ulandelelwano olupheleleyo lwe-transcriptome lubonelela ngendlela ebanzi yokuchaza iimolekyuli ezahlukeneyo ze-RNA, eziquka iikhowudi (mRNA) kunye nee-RNA ezingakhowudi (lncRNA, circRNA, kunye ne-miRNA). Obu buchule bubamba yonke i-transcriptome yeeseli ezithile ngexesha elithile, ivumela ukuqonda okupheleleyo kweenkqubo zeselula. Eyaziwa nangokuthi "ulandelelwano lwe-RNA iyonke," ijolise ekutyhileni uthungelwano lolawulo oluntsonkothileyo kwinqanaba le-transcriptome, evumela uhlalutyo olunzulu olufana ne-RNA ekhuphisanayo ye-endogenous (ceRNA) kunye nohlalutyo oludibeneyo lwe-RNA. Oku kuphawula inyathelo lokuqala elibhekiselele kuphawu olusebenzayo, ngakumbi ekutyhileni uthungelwano lolawulo olubandakanya i-circRNA-miRNA-mRNA-esekwe kunxibelelwano lwe-ceRNA.
