Chromium single cell 3 reagent kits v2

Manufactured by 10x Genomics

Sourced in United States

The Chromium Single Cell 3' Reagent Kits v2 are laboratory equipment used for single-cell gene expression analysis. The kits provide reagents and consumables required for the Chromium Controller instrument to perform single-cell partitioning and library preparation.

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Lab products found in correlation

Chromium controller, by 10x Genomics (11 mentions) Dynabeads myone silane beads, by Thermo Fisher Scientific (6 mentions) Hiseq 3000, by Illumina (5 mentions) Chromium single cell 3 library kit v2, by 10x Genomics (4 mentions) Lightcycler 96 system, by Roche (4 mentions) Spriselect beads, by Beckman Coulter (4 mentions) Chromium single cell 3 reagent kit v2, by 10x Genomics (3 mentions) Hiseq 2500 sequencer, by Illumina (3 mentions) Nextseq 500, by Illumina (3 mentions) Spriselect magnetic beads, by Beckman Coulter (2 mentions) Dynabeads myone silane magnetic beads, by Thermo Fisher Scientific (2 mentions) Agilent 2200 tapestation with high sensitivity d5000 screentape, by Agilent Technologies (2 mentions) Library quantification kit for truseq, by Illumina (2 mentions) Hiseq 2500, by Illumina (2 mentions) Hiseq 4000, by Illumina (2 mentions) Gemcode single cell instrument, by 10x Genomics (2 mentions) Nextseq500 with nextseq 500 550 mid output v2 kit, by Illumina (2 mentions) Agilent 4200 tapestation system, by Agilent Technologies (2 mentions) 35 μm sieve, by BD (2 mentions) Nextera xt dna sample prep kit, by Illumina (2 mentions)

Close spelling variants of this product

Single Cell 3’ Reagent Kit (23 citations) Chromium Single Cell 3′ kit (17 citations) Chromium Single Cell 3’ V2 Kit (6 citations) Chromium Single Cell 3′ v2 (4 citations) Chromium Single Cell Reagent Kit (3 citations) 3′ v2 Reagent Kit (3 citations) 10X Chromium 3’ v2 kit (2 citations)

47 protocols using chromium single cell 3 reagent kits v2

Single-cell RNA-seq of CD45+ PBMCs

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cDNA libraries of CD45⁺ Live PBMCs were generated using the Chromium Single Cell 3ʹ Reagent Kits v2 (10x Genomics) protocol targeting 5,000 cells in two separate wells. Briefly, single cells were isolated into oil emulsion droplets with barcoded gel beads and reverse transcriptase mix. cDNA was generated within these droplets, then the droplets were dissociated. cDNA was purified using DynaBeads MyOne Silane magnetic beads (ThermoFisher, #370002D). cDNA amplification was performed by PCR (10 cycles) using reagents within the Chromium Single Cell 3ʹ Reagent Kit v2 (10x Genomics). Amplified cDNA was purified using SPRIselect magnetic beads (Beckman Coulter). cDNA was enzymatically fragmented and size selected prior to library construction. Libraries were constructed by performing end repair, A-tailing, adaptor ligation, and PCR (12 cycles). Quality of the libraries was assessed by using Agilent 2200 TapeStation with High Sensitivity D5000 ScreenTape (Agilent). Quantity of libraries was assessed by performing digital droplet PCR (ddPCR) with Library Quantification Kit for Illumina TruSeq (BioRad, #1863040). Libraries were diluted to 2 nM and paired-end sequencing was performed on a HiSeq 2500 sequencer (Illumina). The final read depths for the two technical replicates that we sequenced were 77,049 reads/cell and 86,246 reads/cell, respectively.

Mair F., Erickson J.R., Voillet V., Simoni Y., Bi T., Tyznik A.J., Martin J., Gottardo R., Newell E.W, & Prlic M. (2020). A Targeted Multi-omic Analysis Approach Measures Protein Expression and Low-Abundance Transcripts on the Single-Cell Level. Cell reports, 31(1), 107499.

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Single-cell RNA-seq of CD45+ PBMCs

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Single-cell RNA-seq with 10x Genomics

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Libraries prepared with Chromium Single Cell 3′ Reagent Kits v2 (10x Genomics; Cat. #120237) were sequenced on an Illumina HiSeq 2500 (Illumina, San Diego, CA). Cell Ranger pipeline (10x Genomics) was used to convert BCL files into FASTQ files, perform STAR alignment,⁵⁸ (link) filter, count UMIs, and generate gene-barcode matrices. Cell Ranger Aggr pipeline (10x Genomics; v. 3.0.0) was used to aggregate multiple samples, normalize outputs, and recompute gene-barcode matrices on combined data.

Wright C.M., Schneider S., Smith-Edwards K.M., Mafra F., Leembruggen A.J., Gonzalez M.V., Kothakapa D.R., Anderson J.B., Maguire B.A., Gao T., Missall T.A., Howard M.J., Bornstein J.C., Davis B.M, & Heuckeroth R.O. (2021). scRNA-Seq Reveals New Enteric Nervous System Roles for GDNF, NRTN, and TBX3. Cellular and Molecular Gastroenterology and Hepatology, 11(5), 1548-1592.e1.

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Single-cell transcriptome profiling using 10X Genomics

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Single-cell 3′ gene expression libraries were prepared for each sample targeting10,000 cells for cell recovery using the Chromium Single Cell 3′ Reagent Kits v2 and the Chromium single-cell system (10X Genomics, Inc). The resulting library was sequenced using a custom program for 26 bp plus 98 bp paired-end sequencing on a NovaSeq 6000 sequencer (Illumina, Inc, San Diego, CA). Approximately 50,000 reads per cell were generated.

Chen D., Abu Zaid M.I., Reiter J.L., Czader M., Wang L., McGuire P., Xuei X., Gao H., Huang K., Abonour R., Walker B.A, & Liu Y. (2021). Cryopreservation Preserves Cell-Type Composition and Gene Expression Profiles in Bone Marrow Aspirates From Multiple Myeloma Patients. Frontiers in Genetics, 12, 663487.

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Single-cell RNA Sequencing of Renal Endothelial Cells

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Single cell suspensions of freshly isolated renal ECs were resuspended in PBS containing 0.04% ultra-pure BSA. scRNA-seq libraries were prepared using the Chromium Single Cell 3’ Reagent Kits v2 (10x Genomics). The cell recovery for each library was 3000. We used Illumina HiSeq4000 to sequence the libraries, and analyzed the mouse genome using CellRanger (10x Genomics, version 3.1.0). The sample data was then aggregated using the CellRanger software, and the resulting expression matrix was further processed using the Seurat R single cell package (version 4.1.0). Cells with fewer than 500 genes (low quality) or more than 5000 genes (possible doublets), or a mitochondrial gene ratio greater than 5% were excluded from the analysis. In the end, a total of 7,381 renal ECs from young mice and 7,432 renal ECs from old mice with high quality were further analyzed. The data was normalized using the NormalizeData function, followed by the Seurat FindVariableFeatures function as implemented in the Seurat package.

Li M., Wang D., Liu Z., Huang Y., Zhang Q., Pan C., Lin Y., Sun L, & Zheng Y. (2023). Assessing the effects of aging on the renal endothelial cell landscape using single-cell RNA sequencing. Frontiers in Genetics, 14, 1175716.

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Single-cell analysis of NK cells

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NK cells from spleen and peritoneal cavity were isolated after 0, 3 or 7 days of infection, as indicated in Figure 3A. Sorted NK cells (13,000 cells each, cell viability >98%) were encapsulated into droplets, and libraries were prepared using Chromium Single Cell 3’ Reagent Kits v2 according to the manufacturer’s protocol (10X Genomics). The generated scRNA-Seq libraries were sequenced using a 50 cycle (paired-end) with a HiSeq 3000 (Illumina).

Sciumè G., Mikami Y., Jankovic D., Nagashima H., Villarino A.V., Morrison T., Yao C., Signorella S., Sun H.W., Brooks S.R., Fang D., Sartorelli V., Nakayamada S., Hirahara K., Zitti B., Davis F.P., Kanno Y., O’Shea J.J, & Shih H.Y. (2020). Rapid enhancer remodeling and transcription factor repurposing enable high magnitude gene induction upon acute activation of NK cells. Immunity, 53(4), 745-758.e4.

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Single-cell transcriptome profiling using 10x Genomics and BGISEQ-500

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Cells were concentrated to 700–1000 cells/μL and loaded on GemCode Single Cell Instrument (10x Genomics; Pleasanton, CA, USA) to generate single-cell gel bead-in-emulsions (GEMs). Next, GEMs were subjected to library construction using Chromium Single Cell 3’ Reagent Kits v2 (10x Genomics; Pleasanton, CA, USA) according to the manufacturer’s instructions, the steps of which included incubation at room temperature, complementary DNA amplification, fragmentation, end repair, A-tailing, adapter ligation, and sample index polymerase chain reaction. To be compatible with BGISEQ-500 sequencing platform, libraries conversion was performed using the MGIEasy Universal Library Conversion Kit (App-A) (Lot: 1000004155, BGI). Then the converted library was subjected to subsequent DNA circularization and rolling-cycle amplification to generate DNA nanoballs. Purified DNA nanoballs were sequenced using the BGISEQ-500 sequencing platform, generating reads containing 16 base pairs of 10xTM barcodes, 10 base pairs of UMIs, and 100 base pairs of 3’ complementary DNA sequences.

Wang Y., Yu Y., Li L., Zheng M., Zhou J., Gong H., Feng B., Wang X., Meng X., Cui Y., Xia Y., Chu S., Lin L., Chang H., Zhou R., Ma M., Li Z., Ji R., Lu M., Yang X., Zuo X., Li S, & Li Y. (2023). Bile acid-dependent transcription factors and chromatin accessibility determine regional heterogeneity of intestinal antimicrobial peptides. Nature Communications, 14, 5093.

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Single-cell RNA-seq library preparation

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Cellular suspensions were loaded on the Chromium™ Controller (10x Genomics, Pleasanton, CA, USA) in order to generate Gel Bead-In-EMulsions (GEMs). Barcoded sequencing libraries were generated by using the Chromium™ Single Cell 3’ Reagent Kits v2 (10x Genomics, Pleasanton, CA, USA) following manufacturer’s instructions. Sequencing libraries were loaded at 1.4 pM on an Illumina NextSeq500 with NextSeq 500/550 Mid Output v2 kit (150 cycles) (Illumina, CA, USA) using the following read lengths: 26 bp for Read1 (16 bp Barcode + 10 bp Randomer), 8 bp for Sample Index and 58 bp for Read2.

Radermecker C., Sabatel C., Vanwinge C., Ruscitti C., Maréchal P., Perin F., Schyns J., Rocks N., Toussaint M., Cataldo D., Johnston S.L., Bureau F, & Marichal T. (2019). Locally instructed CXCR4hi neutrophils trigger environment-driven allergic asthma through the release of neutrophil extracellular traps. Nature immunology, 20(11), 1444-1455.

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Single-cell RNA-seq of CD8+ T cell subsets

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T cells were isolated as for bulk RNA sequencing, from S. epidermidis-colonized IL-17A-fate-mapping mice, with three groups: Tc1 (Viable Lineage⁻CD45⁺CD90.2⁺CD8β⁺CCR6⁻), Tc17 IL-17A^FM− (Viable Lineage⁻CD45⁺CD90.2⁺CD8β⁺CCR6⁺eYFP⁻) and Tc17 IL-17A^FM+ (Viable Lineage⁻CD45⁺CD90.2⁺CD8β⁺CCR6⁺eYFP⁺). Freshly isolated cells were encapsulated into droplets, and libraries prepared using Chromium Single Cell 3′ Reagent Kits v2 (10X Genomics). The generated scRNA-seq libraries were sequenced using 26 cycles of Read 1, 8 cycles of i7 Index, and 98 cycles of Read2 with a HiSeq 3000 (Illumina).

Harrison O.J., Linehan J.L., Shih H.Y., Bouladoux N., Han S.J., Smelkinson M., Sen S.K., Byrd A.L., Enamorado M., Yao C., Tamoutounour S., Van Laethem F., Hurabielle C., Collins N., Paun A., Salcedo R., O’Shea J.J, & Belkaid Y. (2018). Commensal-specific T cell plasticity promotes rapid tissue adaptation to injury. Science (New York, N.Y.), 363(6422), eaat6280.

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Droplet-based single-nucleus RNA-seq protocol

Cited 1 time

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Droplet-based single-nucleus RNA-sequencing (snRNA-seq) was performed using the Chromium Single Cell 3′ Reagent Kits v2 from 10X Genomics. Nuclei were resuspended to a concentration of 1000 nuclei/uL in 30% Optiprep solution before loading according to manufacturer’s protocol, with 10,000 nuclei recovered per sample as the target. cDNA fragment analysis was performed using the Agilent 4200 TapeStation System. Sequencing parameters and quality control were performed as described by The Tabula Muris Consortium^{59 (link)}.

Leng K., Li E., Eser R., Piergies A., Sit R., Tan M., Neff N., Li S.H., Rodriguez R.D., Suemoto C.K., Leite R.E., Ehrenberg A.J., Pasqualucci C.A., Seeley W.W., Spina S., Heinsen H., Grinberg L.T, & Kampmann M. (2021). Molecular characterization of selectively vulnerable neurons in Alzheimer’s Disease. Nature neuroscience, 24(2), 276-287.

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