Chromium next gem chip k single cell kit

Manufactured by 10x Genomics

Sourced in United States

The Chromium Next GEM Chip K Single Cell Kit is a laboratory equipment product designed for single-cell analysis. It enables the isolation and barcoding of individual cells for further downstream processing and analysis.

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Lab products found in correlation

Chromium next gem single cell 5 kit v2, by 10x Genomics (5 mentions) Novaseq 6000 system, by Illumina (3 mentions) Chromium controller, by 10x Genomics (3 mentions) Chromium single cell human bcr amplification kit, by 10x Genomics (2 mentions) Chromium next gem single cell 3 kit, by 10x Genomics (2 mentions) Chromium next gem single cell 5 kit, by 10x Genomics (2 mentions) Chromium next gem chip g, by 10x Genomics (2 mentions) Qubit 3.0 fluorometer, by Thermo Fisher Scientific (2 mentions) Cg000330 rev c, by 10x Genomics (2 mentions) Sp200 kit, by Illumina (2 mentions) Tapestation, by Agilent Technologies (2 mentions) 5 feature barcode kit, by 10x Genomics (1 mentions) Dual index kit tt set a, by 10x Genomics (1 mentions) Novaseq 6000, by 10x Genomics (1 mentions) Qubit fluorometer, by Thermo Fisher Scientific (1 mentions) Library construction kit, by 10x Genomics (1 mentions) Dual index kit tn set a, by 10x Genomics (1 mentions) Facsdiva 8, by BD (1 mentions) Facsaria 2, by BD (1 mentions) Novaseq s4 flow cell, by Illumina (1 mentions)

Spelling variants of this product

Chromium chip (15 citations) Chromium Next GEM Chip K (7 citations) Chip Kit (6 citations) Chromium chip K (2 citations)

9 protocols using chromium next gem chip k single cell kit

Single-Cell Immune Profiling with Multiplexed Antibodies

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After being processed, the 3 immune cell population fractions were stained with TotalSeq-C Human Universal Cocktail, V1.0 (BioLegend, catalog 399905), as well as Total-Seq anti–human CD72, IgG Fc, CD197 (CCR7), CD45RB, CD193 (CCR3), TCR, LAIR1 (CD395), and CD366 (Tim-3) (BioLegend, catalogs 316211, 410727, 353251, 310211, 310733, 331231, 342807, and 345049, respectively). Cells were pooled and washed using the Laminar Wash Mini system (Curiox Biosystems) before using the Chromium Next GEM Single Cell 5’ Kit v2 and Chromium Next GEM Chip K Single Cell Kit (10X Genomics).
Libraries were created using the Library Construction Kit, 5’Feature Barcode Kit, Chromium Single Cell Human BCR Amplification Kit, Dual Index Kit TT Set A (PN-1000215), and Dual Index Kit TN Set A (PN-1000250) (10X Genomics).Their quality and quantity was assessed using the Agilent Tapestation system and the Qubit Fluorometer (Invitrogen), before they were sent for sequencing on Illumina NovaSeq 6000 as per the instructions provided in 10X Genomics user guide. Sequencing was performed by SNP&SEQ Technology Platform, Science for Life Laboratory (Uppsala Biomedical Centre, Uppsala University, Sweden).

Scharf L., Axelsson H., Emmanouilidi A., Mathew N.R., Sheward D.J., Leach S., Isakson P., Smirnov I.V., Marklund E., Miron N., Andersson L.M., Gisslén M., Murrell B., Lundgren A., Bemark M, & Angeletti D. (2023). Longitudinal single-cell analysis of SARS-CoV-2–reactive B cells uncovers persistence of early-formed, antigen-specific clones. JCI Insight, 8(1), e165299.

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Single-cell 3' and 5' RNA-seq

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Nuclei or cells intended for single-cell/nucleus RNA-seq (8500 nuclei/cells per sample) were directly loaded onto the Chromium Next GEM Chip G or Chromium Next GEM Chip K Single Cell Kit along with the reverse transcription mastermix following the manufacturer’s protocol for the Chromium Next GEM single cell 3′ kit (PN-1000268, 10x Genomics) or Chromium Next GEM Single Cell 5’ Kit (PN-1000263, 10x Genomics), respectively, to generate single-cell gel beads in emulsion. cDNA amplification was done as per the guidelines from 10x Genomics using 13 cycles of amplification for 3′ and 15 cycles of amplification for 5′ libraries. Sequencing libraries were generated with unique dual indices (TT set A) and pooled for sequencing on a Novaseq6000 using a 100-cycle kit and 28-10-10-90 reads.

Garza R., Atacho D.A., Adami A., Gerdes P., Vinod M., Hsieh P., Karlsson O., Horvath V., Johansson P.A., Pandiloski N., Matas-Fuentes J., Quaegebeur A., Kouli A., Sharma Y., Jönsson M.E., Monni E., Englund E., Eichler E.E., Gale Hammell M., Barker R.A., Kokaia Z., Douse C.H, & Jakobsson J. (2023). LINE-1 retrotransposons drive human neuronal transcriptome complexity and functional diversification. Science Advances, 9(44), eadh9543.

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Single-cell transcriptome and BCR profiling

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Synovial tissue samples were thawed and resuspended according to the 10X protocol as described above. Cells were washed and then stained in FACS buffer at 4°C for 20 minutes with fluorescently conjugated antibodies as described in Supplementary Table 3. Single CD19^+ve DAPI^-ve CD3^-ve CD14^-ve B cells were then sorted with the FACS Aria II and FACS Diva 8.0.1 (BD Biosciences). The full gating strategy is shown in Supplementary Figure S2A. Immediately after sorting the CD19^+ve B cells were processed through the Chromium Single Cell Platform using the Chromium Next GEM Single Cell 5’ Kit v2 (10X Genomics, PN- 1000265), the Chromium Single Cell Human BCR Amplification Kit (10X Genomics, PN-1000253), and the Chromium Next GEM Chip K Single Cell Kit (10X Genomics, PN-1000287) as per manufacturer’s protocol. GEX and V(D)J libraries were then sequenced using the NextSeq2000 at the Wellcome Trust Clinical Research Facility’s Genetics Core in Edinburgh.

Wing E., Sutherland C., Miles K., Gray D., Goodyear C.S., Otto T.D., Breusch S., Cowan G, & Gray M. (2023). Double-negative-2 B cells are the major synovial plasma cell precursor in rheumatoid arthritis. Frontiers in Immunology, 14, 1241474.

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Single-cell transcriptomics and TCR profiling

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Single cell transcriptomic libraries were generated using the 5’v2 Gene expression and immune profiling kit (10× Genomics). Viable PBMCs were sorted into 2% FBS/PBS and cell counts were performed using a haemocytometer. Up to 40,000 cells were loaded into each lane of Chromium Next GEM Chip K Single Cell Kit (10× Genomics) to achieve a recovery cell number of approximately 20,000 cells. Subsequent cDNA and TCR libraries were generated according to manufacturer's instructions. Generated libraries were sequenced on the NovaSeq S4 flow cell (Illumina) at Read 1 = 28, i7 index = 10, i5 index = 10 and Read 2: 90 cycles according to manufacturer's instructions.

Khoo W.H., Jackson K., Phetsouphanh C., Zaunders J.J., Alquicira-Hernandez J., Yazar S., Ruiz-Diaz S., Singh M., Dhenni R., Kyaw W., Tea F., Merheb V., Lee F.X., Burrell R., Howard-Jones A., Koirala A., Zhou L., Yuksel A., Catchpoole D.R., Lai C.L., Vitagliano T.L., Rouet R., Christ D., Tang B., West N.P., George S., Gerrard J., Croucher P.I., Kelleher A.D., Goodnow C.G., Sprent J.D., Powell J.E., Brilot F., Nanan R., Hsu P.S., Deenick E.K., Britton P.N, & Phan T.G. (2022). Tracking the clonal dynamics of SARS-CoV-2-specific T cells in children and adults with mild/asymptomatic COVID-19. Clinical Immunology (Orlando, Fla.), 246, 109209.

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High-Throughput Single-Cell VDJ Profiling

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Sorted single cells were segregated into nanoliter droplets (Chromium Next GEM Single Cell 5’ Kit v2, PN-1000263, Chromium Next GEM Chip K Single Cell Kit, PN-1000286, Chromium Controller, 10X Genomics, Pleasanton, CA, USA). The VDJ and antibody feature barcode libraries were made per user guide (CG000330 Rev C, 10X Genomics). Library quality was measured by TapeStation (Agilent, Santa Clara, CA, USA). Library DNA quantification was measured by Qubit 3.0 Fluorometer (ThermoFisher, Waltham, MA, USA). Sequencing depth was 5,000 paired reads per cell with configuration 26, 10, 10, 150, assuming 10,000 cells per library. Sequencing used the NovaSeq 6000 System (Illumina, San Diego, CA, ISA) and SP200 kit (Illumina).

Ford E.S., Mayer-Blackwell K., Jing L., Sholukh A.M., St. Germain R., Bossard E.L., Xie H., Pulliam T.H., Jani S., Selke S., Burrow C.J., McClurkan C.L., Wald A., Holbrook M.R., Eaton B., Eudy E., Murphy M., Postnikova E., Robins H.S., Elyanow R., Gittelman R.M., Ecsedi M., Wilcox E., Chapuis A.G., Fiore-Gartland A, & Koelle D.M. (2022). CD8+ T cell clonotypes from prior SARS-CoV-2 infection predominate during the cellular immune response to mRNA vaccination. Research Square.

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Single-cell 3' and 5' RNA-seq

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Single-Cell Antibody Sequencing

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Sorted single cells were segregated into nanoliter droplets (Chromium Next GEM Single Cell 5' Kit v2, PN-1000263, Chromium Next GEM Chip K Single Cell Kit, PN-1000286, Chromium Controller, 10X Genomics, Pleasanton, CA, USA). The VDJ and antibody feature barcode libraries were made per user guide (CG000330 Rev C, 10X Genomics). Library quality was measured by TapeStation (Agilent, Santa Clara, CA, USA). Library DNA quantification was measured by Qubit 3.0 Fluorometer (ThermoFisher, Waltham, MA, USA). Sequencing depth was 5,000 paired reads per cell with configuration 26, 10, 10, 150, assuming 10,000 cells per library. Sequencing used the NovaSeq 6000 System (Illumina, San Diego, CA, USA) and SP200 kit (Illumina).

Ford E.S., Mayer-Blackwell K., Jing L., Laing K.J., Sholukh A.M., St Germain R., Bossard E.L., Xie H., Pulliam T.H., Jani S., Selke S., Burrow C.J., McClurkan C.L., Wald A., Greninger A.L., Holbrook M.R., Eaton B., Eudy E., Murphy M., Postnikova E., Robins H.S., Elyanow R., Gittelman R.M., Ecsedi M., Wilcox E., Chapuis A.G., Fiore-Gartland A, & Koelle D.M. (2024). Repeated mRNA vaccination sequentially boosts SARS-CoV-2-specific CD8⁺ T cells in persons with previous COVID-19. Nature immunology, 25(1).

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Single-Cell Profiling of Murine Immune Cells

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To prepare samples for scRNA-seq analysis, bone marrow, lymph node and peripheral blood cells isolated from C57BL/6 J female mice were stained with Live/Dead dye (Fixable Viability Stain 510, cat# 564406, BD Pharmingen) and lymphocytes were isolated using a BD FACSAria™ III Sorter (Fig. 1a). Cells were manually counted by Trypan blue (Thermo, T10282) and AO-PI (LUNA, D23001) after each centrifugation and resuspension. Single cells were processed using Chromium Controller (10x Genomics) according to the manufacturer’s protocol. By using Chromium Next GEM Single Cell 5’ Kit v2 (10x Genomics, 1000263) and Chromium Next GEM Chip K Single Cell Kit (10x Genomics, 1000287), we performed single cell TCR/BCR-seq and 5’ gene expression profiling. The cell suspension was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Cell-barcoded 5’ gene expression libraries and V(D)J enriched TCR/BCR libraries were sequenced on an Illumina NovaSeq6000 system by Shanghai Biochip Co., Ltd., Shanghai, China.

zhang Y., Guo C., Zhou Y., Zhang W., Zhu Z., Wang W, & Wan Y. (2024). A biphenotypic lymphocyte subset displays both T- and B-cell functionalities. Communications Biology, 7, 28.

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Characterization of CD8+ T Cells

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Cryopreserved FTD were thawed in RPMI and then stained using the following mAbs: APC anti-human CD3 and FITC anti-human CD8. Zombie NIR Fixable Viability Kit (BioLegend) was used to exclude dead cells. CD3⁺CD8⁺ cells were sorted on an Aria III (BD Biosciences).
GEM (Gel Bead-In EMulsions) generation and barcoding from single-cell suspensions (5000–10,000 CD8⁺ T cells) was performed using the Chromium Next GEM Single Cell 5’ GEM Kit V.2 (PN-1000244) on the Chromium Next GEM Chip K Single Cell Kit (PN-1000286) according to the manufacturer’s instructions (10x Genomics, Pleasanton, California, USA). After GEMs were broken and pooled complementary DNAs (cDNAs) were amplified, TCR target amplification was done using a TCR Amplification Kit (PN-1000252). The TCR and gene expression cDNA libraries were constructed using the Library Construction Kit (PN-1000190). The cDNA quality was assessed using an Agilent 2100 Bioanalyzer system (Agilent, Santa Clara, California, USA) and sequenced using an HiSeq System (Illumina, San Diego, California, USA) with a pair-end 150 bp sequencing strategy.

Komuro H., Shinohara S., Fukushima Y., Demachi-Okamura A., Muraoka D., Masago K., Matsui T., Sugita Y., Takahashi Y., Nishida R., Takashima C., Ohki T., Shigematsu Y., Watanabe F., Adachi K., Fukuyama T., Hamana H., Kishi H., Miura D., Tanaka Y., Onoue K., Onoguchi K., Yamashita Y., Stratford R., Clancy T., Yamaguchi R., Kuroda H., Doi K., Iwata H, & Matsushita H. (2023). Single-cell sequencing on CD8+ TILs revealed the nature of exhausted T cells recognizing neoantigen and cancer/testis antigen in non-small cell lung cancer. Journal for Immunotherapy of Cancer, 11(8), e007180.

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