BMKMANU S3000_Spatial Transcriptome
BMKMANU S3000 Spatial Transcriptome Technical Scheme
Likaroloana
- Qeto: 3.5 µM
- Spot Diameter: 2.5 µM
- Palo ea libaka: hoo e ka bang limilione tse 4
- Lifomate tse 3 tse ka khonehang tsa sebaka sa ho hapa: 6.8 mm * 6.8 mm, 11 mm * 11 mm kapa 15 mm * 20 mm
- Sefaha se seng le se seng se nang le barcode se na le li-primers tse entsoeng ka likarolo tse 4:
• poly(dT) mohatla bakeng sa priming ea mRNA le cDNA synthesis,
• Unique Molecular Identifier (UMI) ho lokisa leeme la ho holisa
• Sebaka sa barcode
• Tatelano e tlamang ea karolo e itseng ea bala 1 ea tatellano
- H&E le litšila tsa fluorescent tsa likarolo
- Monyetla oa ho sebelisa theknoloji ea karohano ea lisele: ho kopanngoa ha litšila tsa H&E, litšila tsa fluorescent, le tatellano ea RNA ho fumana meeli ea sele e 'ngoe le e' ngoe le ho fana ka polelo ea lefutso ka nepo ho sele ka 'ngoe. E sebetsana le tlhahlobo ea profil ea Spatial e theiloeng holim'a sele.
- E khona ho fihlela tlhahlobo ea tharollo ea maemo a mangata: Tlhahlobo e feto-fetohang ea maemo a mangata ho tloha ho 100um ho isa ho 3.5 um ho rarolla likarolo tse fapaneng tsa lisele ka tharollo e nepahetseng.
Melemo ea BMKMANU S3000
-Ho imena habeli ha libaka tsa ho hapa ho fihla ho limilione tse 4: ka qeto e ntlafalitsoeng ea 3.5 uM, e lebisang ho ponelopele e phahameng ea lefutso le UMI ka seleng. Sena se fella ka khokahanyo e ntlafalitsoeng ea lisele tse ipapisitseng le li-profaele tsa mongolo, tse nang le lintlha tse hlakileng haholoanyane tse tsamaellanang le sebopeho sa lisele.
- Qeto ea sub-cellular:Sebaka se seng le se seng sa ho ts'oaroa se ne se e-na le > Li-Barcoded Spots tse limilione tse 2 tse bophara ba 2.5 µm le sebaka sa 5 µm lipakeng tsa litsi tsa libaka, se nolofalletsang tlhahlobo ea transcriptome ea sebaka ka qeto ea sub-cellular (5 µm).
-Tlhahlobo ea tharollo ea maemo a mangata:Tlhahlobo e feto-fetohang ea maemo a mangata ho tloha ho 100 μm ho isa ho 5 μm ho rarolla likarolo tse fapaneng tsa lisele ka tharollo e nepahetseng.
-Monyetla oa ho sebelisa theknoloji ea likarolo tsa "Tse tharo ho slide se le seng":Ho kopanya mabala a fluorescence, H&E staining, le tatellano ea RNA selaeteng se le seng, algorithm ea rona ea tlhahlobo ea "tharo-in-one" e matlafatsa ho tsebahatsa meeli ea lisele bakeng sa li-transcriptomics tse latelang tse thehiloeng liseleng.
-E lumellana le li-platform tse ngata tsa tatellano: ka bobeli NGS le tatellano ea nako e telele e baloang e fumaneha.
-Moralo o feto-fetohang oa sebaka sa 1-8 se ts'oaroang se sebetsang: boholo ba sebaka sa ho hapa sea fetoha, ho khoneha ho sebelisa liforomo tse 3 (6.8 mm * 6.8 mm., 11 mm * 11 mm le 15 mm * 20 mm)
-Tšebeletso e le 'ngoe: e kopanya boiphihlelo bohle le mehato e ipapisitseng le boiphihlelo, ho kenyelletsa le cryo-sectioning, staining, optimization ea lisele, barcoding ea sebaka, ho lokisa laeborari, tatellano, le bioinformatics.
-Bioinformatics e phethahetseng le pono e bonolo ea basebelisi ea liphetho:sephutheloana akarelletsa 29 analysis le 100+ boleng bo phahameng lipalo, ho kopantswe le tshebediso ya inhouse ntshetswa pele Software ho bona ka mahlo a kelello le Customize sele petsoha le letheba clustering.
-Tlhahlobo ea data le pono e ikhethileng: e fumaneha bakeng sa likopo tse fapaneng tsa lipatlisiso
-Sehlopha sa botekgeniki se nang le litsebo tse phahameng: o na le boiphihlelo mefuteng ea linama tse fetang 250 le mefuta e fetang 100+ ho kenyeletsoa batho, litoeba, liphoofolo tse anyesang, litlhapi le limela.
-Lintlafatso tsa nako ea 'nete morerong oohle: ka taolo e felletseng ea tsoelopele ea liteko.
-Tlhahlobo e kopaneng ea boikhethelo ka tatellano ea sele e le 'ngoe ea mRNA
Litlhaloso tsa Tšebeletso
| Litlhoko tsa Mohlala | Laebrari | Leano la Tatelano | Lintlha li khothalelitsoe | Taolo ea Boleng |
| Mehlala ea cryo e kentsoeng ka OCT (Bophara bo lekaneng: approx. 6×6×6mm³) Li-blocks tse 2 ka mohlala 1 bakeng sa liteko, 1 bakeng sa bekapo | S3000 cDNA Library | Illumina PE150 | 160K PE e baloa ka 100υM (250 Gb) | RIN > 7 |
Bakeng sa lintlha tse ling mabapi le tataiso ea boitokisetso ea mohlala le ts'ebetso ea ts'ebeletso, ka kopo ikutloe u lokolohile ho bua le aBMKGENE setsebi
Phallo ea Mosebetsi oa Ts'ebeletso
Mokhahlelong oa boitokiso oa sampole, teko ea pele ea ho ntša RNA ka bongata e etsoa ho netefatsa hore RNA ea boleng bo holimo e ka fumanoa. Mokhahlelong oa ntlafatso ea lithane likarolo li silafatsoa le ho bonoa, 'me maemo a permeabilization bakeng sa tokollo ea mRNA ho tsoa liseleng a ntlafatsoa. Protocol e ntlafalitsoeng e sebelisoa nakong ea kaho ea laeborari, e lateloe ke tatellano le tlhahlobo ea data.
Ts'ebetso e felletseng ea ts'ebeletso e kenyelletsa lintlafatso tsa nako ea nnete le netefatso ea bareki ho boloka sephetho se arabelang, ho netefatsa ts'ebetso ea projeke e bonolo.
Lintlha tse hlahisoang ke BMKMANU S3000 li hlahlojoa ho sebelisoa software "BSTMatrix", e entsoeng ka boithaopo ke BMKGENE, e hlahisang boemo ba sele le tharollo ea mefuta e mengata ea Gene Expression Matrix. Ho tloha moo, ho hlahisoa tlaleho e tloaelehileng e kenyeletsang taolo ea boleng ba data, tlhahlobo ea ka hare-sample le tlhahlobo ea lihlopha tse fapaneng.
- Taolo ea Boleng ba Lintlha:
- Sephetho sa data le kabo ea lintlha tsa boleng
- Ho fumana liphatsa tsa lefutso sebakeng ka seng
- Ho koaheloa ha lisele
- Tlhahlobo ea mohlala e ka hare:
- Leruo la liphatsa tsa lefutso
- Spot clustering, ho kenyelletsa le tlhahlobo e fokolitsoeng ea boholo
- Tlhahlobo ea polelo e fapaneng lipakeng tsa lihlopha: ho tsebahatsa liphatsa tsa lefutso tsa matšoao
- Litlhaloso tse sebetsang le ntlafatso ea liphatsa tsa lefutso tsa matšoao
- Tlhahlobo ea lihlopha tse fapaneng
- Ho kopanngoa hape ha matheba ho tsoa lisampoleng ka bobeli (mohlala, ho kula le ho laola) le ho kopanya hape
- Ho tsebahatsa liphatsa tsa lefutso tsa matšoao bakeng sa sehlopha ka seng
- Litlhaloso tse sebetsang le ntlafatso ea liphatsa tsa lefutso tsa matšoao
- Pontšo e fapaneng ea sehlopha se tšoanang lipakeng tsa lihlopha
Ho feta moo, BMKGene e hlahisitse "BSTViewer" ke sesebelisoa se bonolo sa mosebelisi se nolofalletsang mosebelisi ho bona ka mahlo a kelello polelo ea lefutso le ho bokellana ha lihlopha ka liqeto tse fapaneng.
BMKGene e fana ka lits'ebeletso tsa profiling ea sebaka ka qeto e nepahetseng ea sele e le 'ngoe (e ipapisitseng le cell bin kapa multilevel square bin ho tloha 100um ho isa ho 3.5um).
Lintlha tsa profil ea sebaka ho tsoa likarolong tsa linama ho S3000 slide li sebelitse hantle joalo ka tlase.
Thuto-pale ea 1: Boko ba toeba
Tlhahlobo ea karolo ea boko ba mouse e nang le S3000 e ile ea fella ka ho tsebahatsa ~ lisele tse 94 000, ka tatellano e bohareng ea ~ 2000 liphatsa tsa lefutso ka sele. Qeto e ntlafetseng ea 3.5 uM e ile ea fella ka ho kopanngoa ho hoholo ha lisele ho ipapisitse le lipaterone tsa mongolo, ka lihlopha tsa lisele tse etsisang likarolo tsa boko tse arohaneng. Sena se bonoa habonolo ka ho bona ka mahlo ho ajoa ha lisele tse kopantsoeng joalo ka oligodendrocyte le lisele tsa microglia, tse batlang li fumaneha ka thoko ho taba e putsoa le e tšoeu, ka ho latellana.
Mohlala oa 2: Embryo ea mouse
Tlhahlobo ea karolo ea embryo ea mouse e nang le S3000 e entse hore ho khethoe ~ lisele tse 2200 000, ka tatellano e bohareng ea ~ 1600 liphatsa tsa lefutso ka sele. Qeto e ntlafetseng ea 3.5 uM e ile ea fella ka ho bokellana ho hoholo ha lisele tse thehiloeng ho mekhoa ea mongolo, ka lihlopha tse 12 sebakeng sa leihlo le lihlopha tse 28 sebakeng sa boko.
Ka hare-sample Analysis Cell clustering:
Boitsebiso ba liphatsa tsa lefutso le kabo ea sebaka:
- tharollo e phahameng ea li-sub-cellular: Ha e bapisoa le S1000 slide, sebaka se seng le se seng sa S3000 se nkiloeng se ne se e-na le > Li-Barcoded Spots tse limilione tse 4 tse bophara ba 2.5 µm le sebaka sa 3.5 µm pakeng tsa litsi tsa libaka, e leng se nolofalletsang tlhahlobo ea transcriptome ea sebaka ka tharollo e phahameng ea sub-cellular. (sekoere bin: 3.5 µm).
- Ts'ebetso e phahameng ea ho hapa: ha e bapisoa le S1000 slide, Median_UMI e eketseha ho tloha ho 30% ho isa ho 70%, Median_Gene e eketseha ho tloha 30% ho ea ho 60%
Morero oa chip ea S1000:
Morero oa chip ea S3000:
