V3 single cell reagent kit

Manufactured by 10x Genomics

Sourced in United States

The V3 single-cell reagent kit is a laboratory product offered by 10x Genomics. The kit is designed for use in single-cell analysis workflows. The core function of the kit is to provide the necessary reagents and materials for processing and preparation of single-cell samples.

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Lab products found in correlation

Countess 2, by Thermo Fisher Scientific (6 mentions) Nextseq 550 sequencing platform, by Illumina (2 mentions) Annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (2 mentions) Countess 2 fl automated cell counter, by Thermo Fisher Scientific (2 mentions) Hiseq 4000 platform, by Illumina (2 mentions) Hiseq 4000, by Illumina (2 mentions) Chromium single cell 3 reagent kit v3, by 10x Genomics (1 mentions) Novaseq 6000, by 10x Genomics (1 mentions) Nonacetylated bsa, by New England Biolabs (1 mentions) Novaseq platform, by Illumina (1 mentions) Non acetylated bovine serum albumin, by New England Biolabs (1 mentions)

8 protocols using v3 single cell reagent kit

Single-Cell RNA-Seq of Immune Cells in Cutaneous Melanoma

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MP and CD45⁺ cells sorted from nonadjacent and IM region of 3 CLM specimens (Supplementary Fig. S1A) were subjected to scRNA-seq analysis. Single-cell suspensions were prepared by tissue mincing and enzyme digestion. FACS-sorted cells were resuspended in 0.5 mL PBS 1X plus 0.04% BSA and washed once by centrifugation at 450 rcf for 7 minutes. Cells were then resuspended in 50 μL and counted with an automatic cell counter (Countess II, Thermo Fisher). Approximately 10,000 cells for each sample were loaded into the Chromium Chip B using the Single Cell Reagent Kit v3 (10X Genomics, catalog no. 1000128) for Gel bead Emulsion generation into the Chromium system. Following capture and lysis, cDNA was synthesized and amplified for 14 cycles following the manufacturer's protocol. 50 ng of the amplified cDNA were then used for each sample to construct barcoded sequencing libraries using the Chromium Single Cell 3′ Reagent Kit v3 (10X Genomics, catalog no. 1000128) following the manufacturer's instructions. Sequencing was performed on the NextSeq550 Illumina sequencing platform following manufacturer's instruction for read generation, reaching at least 35,000 reads as mean reads per cell.

Cortese N., Carriero R., Barbagallo M., Putignano A.R., Costa G., Giavazzi F., Grizzi F., Pasqualini F., Peano C., Basso G., Marchini S., Colombo F.S., Soldani C., Franceschini B., Di Tommaso L., Terracciano L., Donadon M., Torzilli G., Kunderfranco P., Mantovani A, & Marchesi F. (2023). High-Resolution Analysis of Mononuclear Phagocytes Reveals GPNMB as a Prognostic Marker in Human Colorectal Liver Metastasis. Cancer Immunology Research, 11(4), 405-420.

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Single-Cell Transcriptional Profiling of Myeloid Cells

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Mononuclear myeloid cells sorted from distal and PT region of three CLM specimens were subjected to single-cell RNA analysis. Single-cell suspensions were prepared by tissue mincing and enzyme digestion. FACS-sorted cells were resuspended in 0.5 ml PBS 1X plus 0.04% BSA and washed once by centrifugation at 450 rcf for 7 min. Cells were then resuspended in 50 μl and counted with an automatic cell counter (ThermoFisher; Countess II). Approximately 10,000 cells of each sample were loaded into one channel of the Chromium Chip B using the Single Cell reagent kit v3 (10X Genomics) for gel bead emulsion generation into the Chromium system. Following capture and lysis, cDNA was synthesized and amplified for 14 cycles following the manufacturer’s protocol. 50 ng of the amplified cDNA was then used for each sample to construct Illumina sequencing libraries. Sequencing was performed on the NextSeq550 Illumina sequencing platform following 10X Genomics' instructions for read generation, reaching at least 35,000 reads as mean reads per cell.

Donadon M., Torzilli G., Cortese N., Soldani C., Di Tommaso L., Franceschini B., Carriero R., Barbagallo M., Rigamonti A., Anselmo A., Colombo F.S., Maggi G., Lleo A., Cibella J., Peano C., Kunderfranco P., Roncalli M., Mantovani A, & Marchesi F. (2020). Macrophage morphology correlates with single-cell diversity and prognosis in colorectal liver metastasis. The Journal of Experimental Medicine, 217(11), e20191847.

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Single-cell RNA sequencing protocol

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From this suspension, 16,000 cells per animal were loaded into one channel of the Chromium system using the v3 single cell reagent kit (10x Genomics, Pleasanton, CA) by the CCHMC Gene Expression Core and these libraries were sequenced as a pool on a Novaseq 6000 in the CCHMC DNA Sequencing Core. Details of scRNAseq analysis pipeline, including packages used and parameters, are found in the Supplemental Materials.

Toth A., Steinmeyer S., Kannan P., Gray J., Jackson C.M., Mukherjee S., Demmert M., Sheak J.R., Benson D., Kitzmiller J., Wayman J.A., Presicce P., Cates C., Rubin R., Chetal K., Du Y., Miao Y., Gu M., Guo M., Kalinichenko V.V., Kallapur S.G., Miraldi E.R., Xu Y., Swarr D., Lewkowich I., Salomonis N., Miller L., Sucre J.S., Whitsett J.A., Chougnet C.A., Jobe A.H., Deshmukh H, & Zacharias W.J. (2022). Inflammatory blockade prevents injury to the developing pulmonary gas exchange surface in preterm primates. Science translational medicine, 14(638), eabl8574.

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Single-cell RNA-seq of Cryopreserved Cells

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Cryopreserved cells were rapidly thawed in a 37 °C water bath and resuspended to concentrations of 1,000 cells/μL in PBS with 0.04% nonacetylated BSA (New England Biolabs). Viability analysis was performed with the Annexin V/Dead Cell Apoptosis Kit (Life Technologies Corporation), and all samples had >90% live cells using the Countess II FL Automated Cell Counter (Thermo Fisher Scientific). Single cells were barcoded with the chromium system using the v3 single-cell reagent kit (10x Genomics). Barcoded libraries were pooled and sequenced on the HiSeq 4000 platform (Illumina) generating 150-base pair paired-end reads.

Voigt A.P., Mulfaul K., Mullin N.K., Flamme-Wiese M.J., Giacalone J.C., Stone E.M., Tucker B.A., Scheetz T.E, & Mullins R.F. (2019). Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration. Proceedings of the National Academy of Sciences of the United States of America, 116(48), 24100-24107.

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Single-cell RNA-seq on NovaSeq

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Approximately 7000 single cells per sample were processed with the Chromium system using the v3 single-cell reagent kit (10× Genomics, San Francisco, CA, USA). Barcoded libraries were pooled and sequenced on the NovaSeq platform (Illumina, San Diego, CA, USA), generating 150 bp paired-end reads as per 10× Genomics recommendations, with >30,000 reads per cell.

Emri E., Cappa O., Kelly C., Kortvely E., SanGiovanni J.P., McKay B.S., Bergen A.A., Simpson D.A, & Lengyel I. (2023). Zinc Supplementation Induced Transcriptional Changes in Primary Human Retinal Pigment Epithelium: A Single-Cell RNA Sequencing Study to Understand Age-Related Macular Degeneration. Cells, 12(5), 773.

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Single-Cell RNA-Seq of Frozen Lung Cells

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Frozen CD326⁻CD31⁻CD45⁺ cells from digested lungs or frozen peripheral blood mononuclear cells were thawed and resuspended in 50 μl at a concentration of 1000 cells/μl. After excluding dead cells, ~20,000 cells (~3 to 4000 cells per animal, barcoded to identify individual animals) were then loaded into one channel of the Chromium system using the v3 Single Cell Reagent Kit (10x Genomics) at the Cincinnati Children’s Hospital Medical Center DNA Sequencing and Genotyping Core. After capture and lysis, complementary DNA (cDNA) was synthesized and amplified as per the manufacturer’s protocol (10x Genomics). The amplified cDNA was used to construct Illumina sequencing libraries that were each sequenced using an Illumina HiSeq 4000. See Supplementary Materials and Methods for full details on the analyses done.

Stevens J., Steinmeyer S., Bonfield M., Peterson L., Wang T., Gray J., Lewkowich I., Xu Y., Du Y., Guo M., Wynn J.L., Zacharias W., Salomonis N., Miller L., Chougnet C., O’Connor D.H, & Deshmukh H. (2022). The balance between protective and pathogenic immune responses to pneumonia in the neonatal lung is enforced by gut microbiota. Science translational medicine, 14(649), eabl3981.

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Single-cell viability and sequencing

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Viability analysis was performed using acridine/propidium iodide staining on the Luna software (Logos Biosystems), and all samples were confirmed to be >95% viable (untreated sample=97% viable and treated sample=98.1% viable). Single cells were barcoded with the chromium system using the V.3 single-cell reagent kit (10X Genomics). Barcoded libraries were pooled and sequenced on the HiSeq 4000 platform (Illumina) generating 150-base pair paired-end reads with a requested target recovery of 10,000 cells. cDNA libraries from the samples were qualitatively checked and passed assessment.

Sabree S.A., Voigt A.P., Blackwell S.E., Vishwakarma A., Chimenti M.S., Salem A.K, & Weiner G.J. (2021). Direct and indirect immune effects of CMP-001, a virus-like particle containing a TLR9 agonist. Journal for Immunotherapy of Cancer, 9(6), e002484.

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Cryopreserved Cell Processing and Sequencing

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Cryopreserved cells were thawed in a 37°C water bath and resuspended in PBS + 0.04% non-acetylated bovine serum albumin (New England Biolabs, Ipswich MA) at a concentration of 1000 cells/μL. Viability analysis on peripheral samples was performed with Annexin V/Dead Cell Apoptosis Kit (Life Technologies Corporation, Eugene OR) with viability > 85% using the Countess II FL Automated Cell Counter (ThermoFisher Scientific, Waltham MA). Single cells were captured and barcoded using the Chromium System with the v3 single cell-reagent kit (10x Genomics, Pleasanton CA). Sequencing was performed on pooled libraries using the Illumina HiSeq 4000 platform (San Diego, CA), generating 150 base pair paired-end reads.

Voigt A., Whitmore S., Flamme-Wiese M., Riker M., Wiley L., Tucker B., Stone E., Mullins R, & Scheetz T. (2019). Molecular Characterization of Foveal versus Peripheral Human Retina by Single-Cell RNA Sequencing. Experimental eye research, 184, 234-242.

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