Cg000238

Manufactured by 10x Genomics

CG000238 is a piece of laboratory equipment produced by 10x Genomics. It is designed to aid in the analysis of biological samples. The core function of this product is to facilitate the processing and preparation of samples for further downstream analysis. No additional details or interpretations can be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

Cg000160, by 10x Genomics (2 mentions) Cryostar nx70, by Thermo Fisher Scientific (1 mentions) Pn 1000184, by 10x Genomics (1 mentions) Eclipse ti2 system, by Nikon (1 mentions) Novoseq 6000 system, by Illumina (1 mentions) Nanozoomer s60, by Hamamatsu Photonics (1 mentions) Cm1950, by Leica (1 mentions) Novaseq 6000, by Illumina (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Visium spatial gene expression kit, by 10x Genomics (1 mentions) Cs2 slide scanner, by Leica (1 mentions) Cytation c10 confocal imaging reader, by Agilent Technologies (1 mentions)

Spelling variants of this product

CG000238 Rev A (2 citations)

4 protocols using cg000238

Visium TO Slide Preparation

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Seven OCT/collagen I blocks, frozen as described above, were carefully placed on the seven capture areas of the Visium TO slide while on dry ice. The slide containing the OCT-frozen collagen I layer was thawed at room temperature, and the OCT compound was carefully rinsed off with PBS. The slide was dried for 5 min on the 10x Genomics thermocycler adapter, placed on a thermocycler set to 37°C, and fixed in methanol for 5 min. The TO slide was directly H&E-stained according to the Visium H&E Staining guide (10x Genomics, CG000160) but excluding the methanol fixation step and imaged on an EVOS M7000 system (Invitrogen) using the 10× objective and on the bright-field channel. Immediately after imaging, the slide was processed according to the Visium TO user guide (10x Genomics, CG000238), and the fluorescence signal was imaged using the EVOS M7000 system using the 10× objective and red fluorescent protein channel.

Auxillos J., Crouigneau R., Li Y.F., Dai Y., Stigliani A., Tavernaro I., Resch-Genger U., Sandelin A., Marie R, & Pedersen S.F. (2024). Spatially resolved analysis of microenvironmental gradient impact on cancer cell phenotypes. Science Advances, 10(18), eadn3448.

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Spatial Transcriptomics of DCIS Samples

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Fresh tissues from the two DCIS patients were cut to proper size and embedded in cryomold (Fisher #NC9542860) by OCT compound (Fisher #1437365) on dry ice and stored in −80°C in sealed bags. Frozen OCT embedded DCIS cryosections were cut to 12μm in the cryostat (Thermo Scientific Cryostar NX70) with specimen head temperature at −17 °C and blade temperature at −15 °C. The cut sections were placed within a capture area of the Visium spatial slide (10X Genomics PN-1000184). The slide was permeabilized for 12 minutes according to the Visium Spatial Tissue Optimization protocol (10X Genomics CG000238). Imaging of the stained slides was performed on the Nikon Eclipse Ti2 system. Finally, the ST libraries were constructed by following the Visium Spatial Gene Expression protocol (10X Genomics CG000239) and sequenced at 200 cycles by S1 flowcell on the Novoseq 6000 system (Illumina).

Wei R., He S., Bai S., Sei E., Hu M., Thompson A., Chen K., Krishnamurthy S, & Navin N.E. (2022). Spatial charting of single cell transcriptomes in tissues. Nature biotechnology, 40(8), 1190-1199.

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Spatial Transcriptomics of Tonsil Tissue

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Spatial visualization of gene expression within tonsil tissue was conducted using the Visium Spatial Gene Expression kit (10X Genomics) as per manufacturer's protocol. The OCT blocks were cut twice using a cryostat (Leica CM1950): a first time to assess RNA quality and assure a minimum RNA Integrity Number (RIN) number of 7 (RNA pico Chip) and a second time to mount a 10 μm section on the Visium slides. Slides were H&E stained before the sections were imaged using the NanoZoomer S60 (Hamamatsu) to assess tissue morphology and quality. The sections were then permeabilized for 6 min, according to the results of a corresponding Tissue Optimization experiment (10X Genomics, CG000238), and processed according to the Visium Spatial Gene Expression user guide (10X Genomics, CG000239). In short, tissue was lysed and reverse transcription was performed followed by second strand synthesis and cDNA denaturation. Spatially barcoded, full length cDNAs were amplified by PCR for 16 or 18 cycles, depending on the initial concentration previously determined by qPCR. Indexed sequencing libraries were generated via end repair, A-tailing, adaptor ligation and sample index PCR and analyzed using the Agilent 2100 BioAnalyzer. Libraries were sequenced on an Illumina NovaSeq 6000 with sequencing depth of ∼100,000 reads per spot.

Massoni-Badosa R., Aguilar-Fernández S., Nieto J.C., Soler-Vila P., Elosua-Bayes M., Marchese D., Kulis M., Vilas-Zornoza A., Bühler M.M., Rashmi S., Alsinet C., Caratù G., Moutinho C., Ruiz S., Lorden P., Lunazzi G., Colomer D., Frigola G., Blevins W., Romero-Rivero L., Jiménez-Martínez V., Vidal A., Mateos-Jaimez J., Maiques-Diaz A., Ovejero S., Moreaux J., Palomino S., Gomez-Cabrero D., Agirre X., Weniger M.A., King H.W., Garner L.C., Marini F., Cervera-Paz F.J., Baptista P.M., Vilaseca I., Rosales C., Ruiz-Gaspà S., Talks B., Sidhpura K., Pascual-Reguant A., Hauser A.E., Haniffa M., Prosper F., Küppers R., Gut I.G., Campo E., Martin-Subero J.I, & Heyn H. (2024). An atlas of cells in the human tonsil. Immunity, 57(2), 379-399.e18.

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Optimized Visium Spatial Transcriptomics

Cited 1 time

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Visium Spatial Gene Expression slides were processed as previously described [4 (link),5 (link)]. To ensure the optimal time duration of exposure to permeabilization enzyme, tissue optimization experiments were performed according to the manufacturer’s protocols (protocol CG000160, revision B, 10x Genomics). Tissue sections were scored to include the dentate gyrus to facilitate orientation, and exposed to permeabilization enzyme for differing time durations. cDNA synthesis was performed using a fluorescently-labeled nucleotide (CG000238, revision D, 10x Genomics). The slide was then coverslipped and fluorescent images were acquired at 10x magnification with a TRITC filter (ex 550nm/em 600nm) on a Cytation C10 Confocal Imaging Reader (Agilent). Following this experiment, 18 minutes was selected as the optimal permeabilization time. For each Visium slide, H&E staining was performed (protocol CG000160, revision B, 10x Genomics), after which slides were coverslipped and high-resolution, brightfield images were acquired on a Leica CS2 slide scanner equipped with a 20x/0.75NA objective and a 2x doubler.

Nelson E.D., Tippani M., Ramnauth A.D., Divecha H.R., Miller R.A., Eagles N.J., Pattie E.A., Kwon S.H., Bach S.V., Kaipa U.M., Yao J., Kleinman J.E., Collado-Torres L., Han S., Maynard K.R., Hyde T.M., Martinowich K., Page S.C, & Hicks S.C. (2024). An integrated single-nucleus and spatial transcriptomics atlas reveals the molecular landscape of the human hippocampus. bioRxiv.

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