BMKMANU S3000_Spatial Transcriptome
BMKMANU S3000 Spatial Transcriptome Technical Scheme
Mawonekedwe
- Kusamvana: 3.5 μM
- Spot Diameter: 2.5 µM
- Chiwerengero cha malo: pafupifupi 4 Miliyoni
- 3 zotheka kulanda malo akamagwiritsa: 6.8 mm * 6.8 mm, 11 mm * 11 mm kapena 15 mm * 20 mm
- Mkanda uliwonse wokhala ndi barcode umadzaza ndi zoyambira zopangidwa ndi magawo 4:
• poly(dT) mchira wa mRNA priming ndi cDNA synthesis,
• Unique Molecular Identifier (UMI) kuti mukonze kukondera kokulirakulira
• Barcode ya malo
• Kumangirira koyambira pang'onopang'ono kuwerenga 1 koyambira
- H&E ndi utoto wa fulorosenti wa zigawo
- Kuthekera kogwiritsa ntchito ukadaulo wogawa ma cell: kuphatikiza madontho a H&E, utoto wa fulorosenti, ndi kutsatizana kwa RNA kuti mudziwe malire a selo lililonse ndikugawa molondola mawonekedwe a jini ku selo lililonse. Kukonza kusanthula kwa mbiri ya Spatial kutengera ma cell bin.
- Zotheka kuti mukwaniritse kusanthula kwamitundu yambiri: Kusanthula kosinthika kwamitundu ingapo kuyambira 100um mpaka 3.5 um kuti muthane ndi mitundu yosiyanasiyana ya minofu pakuwongolera koyenera.
Ubwino wa BMKMANU S3000
-Kuwirikiza kawiri malo olanda kufika 4 Miliyoni: ndi kusamvana kwabwino kwa 3.5 uM, zomwe zimatsogolera ku jini yapamwamba ndi kuzindikira kwa UMI pa selo. Izi zimabweretsa kusanjika bwino kwa ma cell kutengera mbiri yolembedwa, yokhala ndi tsatanetsatane wofananira ndi kapangidwe ka minofu.
- Kusintha kwa ma sub-cellular:Malo aliwonse olandirira amakhala ndi Madontho a Barcoded 2 miliyoni okhala ndi mainchesi a 2.5 µm ndi malo otalikirana 5 µm pakati pa malo omwe amawonekera, zomwe zimathandiza kusanthula kwa malo okhala ndi ma sub-cellular resolution (5 µm).
-Kusanthula kwamagawo angapo:Kusanthula kosinthika kwamagawo angapo kuyambira 100 μm mpaka 5 μm kuti athetse mawonekedwe osiyanasiyana a minofu pakuwongolera koyenera.
-Kuthekera kogwiritsa ntchito ukadaulo wa magawo a "Atatu mu slide imodzi":kuphatikiza madontho a fluorescence, madontho a H&E, ndi kutsatizana kwa RNA pa slide imodzi, algorithm yathu yowunikira "atatu-in-one" imathandizira kuzindikira malire a cell a ma cell-based transcriptomics.
-Zimagwirizana ndi mapulatifomu angapo otsatizana: onse NGS ndi kutsata kwanthawi yayitali komwe kulipo.
-Mapangidwe osinthika a 1-8 yogwira ntchito: kukula kwa malo ogwidwa ndi osinthika, zotheka kugwiritsa ntchito mawonekedwe a 3 (6.8 mm * 6.8 mm., 11 mm * 11 mm ndi 15 mm * 20 mm)
-Utumiki woyima kamodzi: imaphatikiza zochitika zonse ndi masitepe otengera luso, kuphatikiza cryo-sectioning, staining, kukhathamiritsa kwa minofu, barcoding ya malo, kukonzekera laibulale, kutsatizana, ndi bioinformatics.
-Ma bioinformatics ndi mawonekedwe osavuta kugwiritsa ntchito:Phukusi limaphatikizapo kusanthula kwa 29 ndi ziwerengero zapamwamba za 100+, kuphatikiza kugwiritsa ntchito mapulogalamu opangidwa m'nyumba kuti muwone ndikusintha mwamakonda kugawanika kwa ma cell ndi kusanja mawanga.
-Kusanthula kwa data ndi makonda: kupezeka pazofunsitsa zosiyanasiyana
-Gulu laukadaulo laluso kwambiri: Wodziwa zambiri pamitundu yopitilira 250 ya minofu ndi mitundu 100+ kuphatikiza anthu, mbewa, zoyamwitsa, nsomba ndi zomera.
-Zosintha zenizeni pa polojekiti yonse: ndi ulamuliro wonse wa kupita patsogolo kwa kuyesera.
-Kusanthula kophatikizana kosankha ndi katsatidwe ka One-cell mRNA
Mafotokozedwe a Utumiki
| Zitsanzo Zofunika | Library | Njira Yotsatirira | Deta Yovomerezeka | Kuwongolera Kwabwino |
| Zitsanzo za cryo zophatikizidwa ndi OCT (Mulingo woyenera kwambiri: pafupifupi. 6 × 6 × 6 mm³) 2 midadada pa chitsanzo 1 yoyesera, 1 yosunga zosunga zobwezeretsera | S3000 cDNA Library | Chithunzi cha PE150 | 160K PE imawerengedwa pa 100υM (250 Gb) | RIN> 7 |
Kuti mumve zambiri zachitsogozo chokonzekera zitsanzo ndi kayendetsedwe ka ntchito, chonde omasuka kulankhula ndi aBMKGENE katswiri
Kuyenda kwa Ntchito Yantchito
Mu gawo lokonzekera lachitsanzo, kuyesa koyambirira kwa RNA kochulukira kumachitika kuti zitsimikizire kuti RNA yapamwamba ingapezeke. Mu gawo la kukhathamiritsa kwa minofu zigawozo zimadetsedwa ndikuwonetseredwa komanso momwe permeabilization yotulutsa mRNA kuchokera ku minofu imakonzedwa. Protocol yokhathamiritsa imagwiritsidwa ntchito pomanga laibulale, ndikutsatiridwa ndi kutsata ndi kusanthula deta.
Kuyenda kwathunthu kwautumiki kumaphatikizapo zosintha zenizeni zenizeni ndi kutsimikizika kwamakasitomala kuti mukhalebe ndi mayankho omvera, kuwonetsetsa kuti polojekiti ichitike bwino.
Deta yopangidwa ndi BMKMANU S3000 imawunikidwa pogwiritsa ntchito pulogalamu ya "BSTMatrix", yomwe imapangidwa paokha ndi BMKGENE, ikupanga mulingo wa cell ndi ma multilevel resolution Gene Expression Matrix. Kuchokera pamenepo, lipoti lokhazikika limapangidwa lomwe limaphatikizapo kuwongolera khalidwe la deta, kusanthula kwachitsanzo chamkati ndi kusanthula magulu.
- Kuwongolera Ubwino wa Data:
- Kutulutsa kwa data ndi kugawa kwamagulu abwino
- Kuzindikira kwa majini pa malo
- Kufalikira kwa minofu
- Kusanthula kwachitsanzo chamkati:
- Kulemera kwa gene
- Kuphatikizika kwamadontho, kuphatikiza kusanthula kwamiyendo
- Kusanthula kwamaganizidwe osiyanasiyana pakati pa magulu: kuzindikiritsa jini zolembera
- Kafotokozedwe kogwira ntchito ndikulemeretsa kwa jini zolembera
- Kusanthula kwamagulu
- Kuphatikizanso mawanga kuchokera ku zitsanzo zonse (monga matenda ndi kuwongolera) ndikuphatikizanso
- Kuzindikiritsa jini zolembera pagulu lililonse
- Kafotokozedwe kogwira ntchito ndikulemeretsa kwa jini zolembera
- Kusiyanitsa kwa gulu limodzi pakati pa magulu
Kuphatikiza apo, BMKGene idapanga "BSTViewer" ndi chida chosavuta kugwiritsa ntchito chomwe chimathandiza wogwiritsa ntchito kuwona m'maganizo amtundu wa jini ndikuphatikizana pazosankha zosiyanasiyana.
BMKGene imapereka ntchito zowunikira pakanthawi kochepa ka cell imodzi (kutengera cell bin kapena multilevel square bin kuyambira 100um mpaka 3.5um).
Deta ya mbiri ya malo kuchokera kumagulu a minofu pa S3000 slide idachita bwino monga pansipa.
Phunziro 1: Ubongo wa mbewa
Kusanthula kwa gawo la ubongo wa mbewa ndi S3000 kunapangitsa kuti ~ 94 000 maselo adziwike, ndi kutsatizana kwapakati kwa ~ 2000 majini pa selo. Kuwongolera kwabwino kwa 3.5 uM kudapangitsa kuti ma cell asakanizidwe mwatsatanetsatane motengera zolemba, ndi magulu a maselo omwe amatsanzira momwe ubongo umapangidwira. Izi zimawonedwa mosavuta powonera kugawidwa kwa maselo ophatikizidwa ngati oligodendrocyte ndi ma cell a microglia, omwe amakhala pafupifupi mu imvi ndi yoyera, motsatana.
Phunziro 2: Mluza wa mbewa
Kuwunika kwa gawo la embryo la mbewa ndi S3000 kudapangitsa kuti ~ 2200 000 maselo adziwike, ndikutsatizana kwapakati kwa ~ 1600 majini pa selo. Kusintha kwabwino kwa 3.5 uM kudapangitsa kuti ma cell asakanidwe mwatsatanetsatane potengera zolemba, ndi masango 12 m'dera la diso ndi masango 28 m'dera la ubongo.
Kuphatikizika kwa Ma cell a Zitsanzo zamkati:
Kuzindikiritsa ma jini a chikhomo ndi kugawa kwamalo:
- mawonekedwe apamwamba a sub-cellular: Poyerekeza ndi slide ya S1000, malo aliwonse ojambulidwa a S3000 amakhala ndi >4 miliyoni ma Barcoded Spots okhala ndi mainchesi a 2.5 µm ndi mtunda wa 3.5 µm pakati pa malo owonekera, kupangitsa kusanthula kwapang'onopang'ono kwapang'onopang'ono (square bin: 3.5 µm).
- Kujambula bwino kwambiri: poyerekeza ndi S1000 slide, Median_UMI ikukwera kuchoka pa 30% mpaka 70%, Median_Gene ikukwera kuchokera 30% mpaka 60%
Ndondomeko ya S1000 chip:
Chip cha S3000:
