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Chromium chip

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The Chromium chip is a core component of the Chromium System, a microfluidics-based platform developed by 10x Genomics for single-cell and spatial analysis. The Chromium chip serves as the processing chamber where individual cells or nuclei are encapsulated into nanoliter-scale gel beads, enabling parallel sequencing of genetic material from thousands of single cells or spatial features.

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Lab products found in correlation

Nextseq 500, by Illumina (5 mentions) Novaseq 6000, by Illumina (4 mentions) Cellranger software v3, by 10x Genomics (3 mentions) Totalseq, by BioLegend (2 mentions) Smart seq2 protocol, by Illumina (2 mentions) Single cell 3 kit, by 10x Genomics (2 mentions) Hiseq 4000 system, by Illumina (1 mentions) Kapa library quant, by Roche (1 mentions) Bioanalyzer, by Agilent Technologies (1 mentions) Qubit dsdna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions) Sequencer, by Illumina (1 mentions) Bioanalyzer high sensitivity dna chip, by Agilent Technologies (1 mentions) Chromium controller, by 10x Genomics (1 mentions) Hiseq platform, by Illumina (1 mentions) Countess 2 automated cell counter, by Thermo Fisher Scientific (1 mentions) Influx cytometer, by BD (1 mentions) Hiseq 2000, by Illumina (1 mentions) Pe conjugated antibody, by BioLegend (1 mentions) Cell ranger v2, by 10x Genomics (1 mentions)

15 protocols using chromium chip

1

Single-Cell B-Cell Receptor Sequencing

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For paired antibody variable gene sequence analysis, cells were resuspended into DPBS containing 0.04% non-acetylated BSA, split into four replicates, and separately added to 50 μL of RT Reagent Mix, 5.9 μL of Poly-dt RT Primer, 2.4 μL of Additive A and 10 μL of RT Enzyme Mix B to complete the Reaction Mix as per the vendor’s protocol. The reactions then were loaded onto a Chromium chip (10x Genomics). Chromium Single Cell V(D)J B-Cell-enriched libraries were generated, quantified, normalized and sequenced according to the User Guide for Chromium Single Cell V(D)J Reagents kit (CG000086_REV C). Amplicons were sequenced on an Illumina Novaseq 6000, and data were processed using the CellRanger software v3.1.0 (10x Genomics).
(1996). Proceedings of the National Academy of Sciences of the United States of America, 93(25), 14993.
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2

Paired Antibody Variable Region Sequencing

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For paired antibody variable region gene sequencing, cells were resuspended into DPBS containing 0.04% non-acetylated bovine serum albumin (BSA), split into four replicates, and separately added to 50 μL of RT Reagent Mix, 5.9 μL of Poly-dt RT Primer, 2.4 μL of Additive A and 10 μL of RT Enzyme Mix B to complete the Reaction Mix, as per the vendor’s protocol. The reactions then were loaded onto a Chromium chip (10x Genomics). Chromium Single Cell V(D)J B-Cell-enriched libraries were generated, quantified, normalized and sequenced according to the User Guide for Chromium Single Cell V(D)J Reagents kits (CG000086_REV C). Amplicons were sequenced on an Illumina Novaseq 6000, and data were processed using the CellRanger software v3.1.0 (10X Genomics). Bioinformatics filtering steps were performed as described previously (26 (link)). The identities of gene segments and CDRs from germlines were determined by alignment using the ImMunoGeneTics database (30 (link)).
Gilchuk P., Guthals A., Bonissone S.R., Shaw J.B., Ilinykh P.A., Huang K., Bombardi R.G., Liang J., Grinyo A., Davidson E., Chen E.C., Gunn B.M., Alter G., Saphire E.O., Doranz B.J., Bukreyev A., Zeitlin L., Castellana N, & Crowe JE J.r. (2021). Proteo-Genomic Analysis Identifies Two Major Sites of Vulnerability on Ebolavirus Glycoprotein for Neutralizing Antibodies in Convalescent Human Plasma. Frontiers in Immunology, 12, 706757.
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3

Optimized scRNA-seq and Bulk RNA-seq Workflow

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For scRNAseq experiments, viable cells in single cell suspension were resuspended in 0.4% BSA in PBS at a concentration of 1,000 cells per ul. 7,000 cells were loaded onto a single lane (Chromium chip, 10X Genomics) followed by encapsulation in lipid droplet (Single Cell 3’ kit, 10X Genomics) followed by cDNA and library generation per manufacturer protocol. For multiplexed scRNAseq experiments32 , cells with stained with cell hashing antibodies (TotalSeq, Biolegend) prior to cell capture. cDNA libraries were sequenced to an average of 50,000 reads per cell using Illumina Nextseq 500. scRNA-seq reads were processed with Cell Ranger v2.1, which demultiplexed cells from different samples and quantified transcript counts per putative cell. Quantification was performed using the STAR aligner against the hg19 transcriptome. For bulk RNA-sequencing experiments, full-length cDNA and sequencing libraries were performed using Illumina Smart-seq2 protocol 33 . Libraries were sequenced on MiSeq from Illumina to generate 35 base paired-end reads. Reads were mapped to the hg19 transcriptome using kallisto 0.42.4 and transcriptional levels of genes were quantified with the Log2(TPM+1) (Transcripts Per Kilobase Million) metric.
Wei K., Korsunsky I., Marshall J.L., Gao A., Watts G.F., Major T., Croft A.P., Watts J., Blazar P., Lange J., Thornhill T., Filer A., Raza K., Donlin L.T., Siebel C.W., Buckley C.D., Raychaudhuri S, & Brenner M.B. (2020). Notch signaling drives synovial fibroblast identity and arthritis pathology. Nature, 582(7811), 259-264.
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4

Single-Cell RNA-Seq Profiling of Diverse Tissues

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Single-cell RNA-sequencing experiments for lung, intestine, and synovium samples were performed through the Brigham and Women’s Hospital Single Cell Genomics Core. Viable cells in single-cell suspension were resuspended in 0.4% BSA in PBS at a concentration of 1,000 cells per ul. 7,000 cells were loaded onto a single lane (Chromium chip, 10X Genomics) followed by encapsulation in lipid droplet, with the 10× Genomics Single-Cell 3′ kit (Version 2 for synovium and intestine, Version 3 for lung) followed by cDNA and library generation per manufacturer protocol. cDNA libraries were sequenced to an average of 50,000 reads per cell using Illumina Nextseq 500. Single-cell RNA-sequencing experiments for salivary gland samples were performed at Oxford University. For each library, 10,000 cells were counted using the automated cell counter Bio-Rad TC20 and loaded onto a single 10× lane and processed with the 10× Genomics Single Cell 3′ kit (Version 3). Sequencing was done using Illumina NovaSeq 6000 and libraries were sequenced to a minimum of 50000 reads/cell.
Korsunsky I., Wei K., Pohin M., Kim E.Y., Barone F., Major T., Taylor E., Ravindran R., Kemble S., Watts G.F., Jonsson A.H., Jeong Y., Athar H., Windell D., Kang J.B., Friedrich M., Turner J., Nayar S., Fisher B.A., Raza K., Marshall J.L., Croft A.P., Tamura T., Sholl L.M., Vivero M., Rosas I.O., Bowman S.J., Coles M., Frei A.P., Lassen K., Filer A., Powrie F., Buckley C.D., Brenner M.B, & Raychaudhuri S. (2022). Cross-tissue, single-cell stromal atlas identifies shared pathological fibroblast phenotypes in four chronic inflammatory diseases. Med (New York, N.Y.), 3(7), 481-518.e14.
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5

Single-Cell RNA-Seq of Stressed Cells

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Cell suspensions with approximately 1,000,000 cells/μl were used. Each pool was loaded onto individual lanes of a 10X Genomics Chromium chip, as per factory recommendations. For all three tissues, the control and stress cell suspensions were loaded and processed together in the same chip to avoid batch effects by condition. Reverse transcription and library preparation were performed using the 10X Genomics Single-Cell v2.0 kit following the 10X Genomics protocol. Molar concentration and fragment length of libraries were quantified by qPCR using KAPA Library Quant (Kapa Biosystems) and Bioanalyzer (Agilent High Sensitivity DNA kit), respectively. Each library was sequenced on a single lane of an Illumina HiSeq4000 System generating 100–base pair paired-end reads at a depth of ~340 million reads per sample.
Lopez J.P., Brivio E., Santambrogio A., De Donno C., Kos A., Peters M., Rost N., Czamara D., Brückl T.M., Roeh S., Pöhlmann M.L., Engelhardt C., Ressle A., Stoffel R., Tontsch A., Villamizar J.M., Reincke M., Riester A., Sbiera S., Fassnacht M., Mayberg H.S., Craighead W.E., Dunlop B.W., Nemeroff C.B., Schmidt M.V., Binder E.B., Theis F.J., Beuschlein F., Andoniadou C.L, & Chen A. (2021). Single-cell molecular profiling of all three components of the HPA axis reveals adrenal ABCB1 as a regulator of stress adaptation. Science Advances, 7(5), eabe4497.
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6

Single-Cell RNA-Seq Library Preparation

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For each sample, 8,000–16,500 nuclei were loaded in one channel of a Chromium Chip (10x Genomics). 3’ v3.1 chemistry was used to process all other tissues. cDNA and gene expression libraries were generated according to the manufacturer’s instructions (10x Genomics). cDNA and gene expression library fragment sizes were assessed with a DNA High Sensitivity Bioanalyzer Chip (Agilent). cDNA and gene expression libraries were quantified using the Qubit dsDNA High Sensitivity assay kit (ThermoFisher Scientific). Gene expression libraries were multiplexed and sequenced on an Illumina sequencer.
Pita-Juarez Y., Karagkouni D., Kalavros N., Melms J.C., Niezen S., Delorey T.M., Essene A.L., Brook O.R., Pant D., Skelton-Badlani D., Naderi P., Huang P., Pan L., Hether T., Andrews T.S., Ziegler C.G., Reeves J., Myloserdnyy A., Chen R., Nam A., Phelan S., Liang Y., Amin A.D., Biermann J., Hibshoosh H., Veregge M., Kramer Z., Jacobs C., Yalcin Y., Phillips D., Slyper M., Subramanian A., Ashenberg O., Bloom-Ackermann Z., Tran V.M., Gomez J., Sturm A., Zhang S., Fleming S.J., Warren S., Beechem J., Hung D., Babadi M., Padera RF J.r., MacParland S.A., Bader G.D., Imad N., Solomon I.H., Miller E., Riedel S., Porter C.B., Villani A.C., Tsai L.T., Hide W., Szabo G., Hecht J., Rozenblatt-Rosen O., Shalek A.K., Izar B., Regev A., Popov Y., Jiang Z.G, & Vlachos I.S. (2022). A single-nucleus and spatial transcriptomic atlas of the COVID-19 liver reveals topological, functional, and regenerative organ disruption in patients. bioRxiv.
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7

Single-cell Antibody Profiling of E-specific B Cells

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The Chromium Single Cell V(D)J workflow with B-cell only enrichment option was chosen for generating linked heavy-chain and light-chain antibody profiling libraries. Approximately 800 directly sorted E-specific B cells were split evenly into two replicates and separately added to 50 μL of RT Reagent Mix, 5.9 μL of Poly-dt RT Primer, 2.4 μL of Additive A and 10 μL of RT Enzyme Mix B to complete the Reaction Mix as per the vendor’s protocol, which then was loaded directly onto a Chromium chip (10x Genomics). Similarly, for the remaining sorted cells that were bulk expanded, approximately 40,000 cells from two separate sorting approaches were split evenly across six reactions and separately processed as described above before loading onto a Chromium chip. The libraries were prepared following the User Guide for Chromium Single Cell V(D)J Reagents kits (CG000086_REV C).
Gilchuk P., Bombardi R.G., Erasmus J.H., Tan Q., Nargi R., Soto C., Abbink P., Suscovich T.J., Durnell L.A., Khandhar A., Archer J., Liang J., Fouch M.E., Davidson E., Doranz B.J., Jones T., Larson E., Ertel S., Granger B., Fuerte-Stone J., Roy V., Broge T., Linnekin T.C., Linde C.H., Gorman M.J., Nkolola J., Alter G., Reed S.G., Barouch D.H., Diamond M.S., Crowe JE J.r., Van Hoeven N., Thackray L, & Carnahan R. (2020). Integrated pipeline for the accelerated discovery of antiviral antibody therapeutics. Nature biomedical engineering, 4(11), 1030-1043.
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8

Profiling SARS-CoV-2 Antibody Responses

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As an alternative approach, we also used a second major approach for isolation of SARS-CoV-2-reactive antibodies. In some experiments, the Chromium Single Cell V(D)J workflow with B-cell only enrichment option was used for generating linked heavy-chain and light-chain antibody profiling libraries. Approximately 2,866 directly sorted S2Pecto or 1,626 RBD-mFc protein-specific B cells were split evenly into two replicates each and separately added to 50 μL of RT Reagent Mix, 5.9 μL of Poly-dt RT Primer, 2.4 μL of Additive A and 10 μL of RT Enzyme Mix B to complete the Reaction Mix as per the vendor’s protocol, which then was loaded directly onto a Chromium chip (10x Genomics). Similarly, for the remaining sorted cells that were expanded in bulk, approximately 40,000 cells from two separate sorting approaches were split evenly across four reactions and processed separately as described above before loading onto a Chromium chip. The libraries were prepared following the User Guide for Chromium Single Cell V(D)J Reagents kits (CG000086_REV K).
Zost S.J., Gilchuk P., Chen R.E., Case J.B., Reidy J.X., Trivette A., Nargi R.S., Sutton R.E., Suryadevara N., Chen E.C., Binshtein E., Shrihari S., Ostrowski M., Chu H.Y., Didier J.E., MacRenaris K.W., Jones T., Day S., Myers L., Lee F.E., Nguyen D.C., Sanz I., Martinez D.R., Rothlauf P.W., Bloyet L.M., Whelan S.P., Baric R.S., Thackray L.B., Diamond M.S., Carnahan R.H, & Crowe JE J.r. (2020). Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein. Nature medicine, 26(9), 1422-1427.
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9

Single-cell Antibody Profiling of E-specific B Cells

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The Chromium Single Cell V(D)J workflow with B-cell only enrichment option was chosen for generating linked heavy-chain and light-chain antibody profiling libraries. Approximately 800 directly sorted E-specific B cells were split evenly into two replicates and separately added to 50 μL of RT Reagent Mix, 5.9 μL of Poly-dt RT Primer, 2.4 μL of Additive A and 10 μL of RT Enzyme Mix B to complete the Reaction Mix as per the vendor’s protocol, which then was loaded directly onto a Chromium chip (10x Genomics). Similarly, for the remaining sorted cells that were bulk expanded, approximately 40,000 cells from two separate sorting approaches were split evenly across six reactions and separately processed as described above before loading onto a Chromium chip. The libraries were prepared following the User Guide for Chromium Single Cell V(D)J Reagents kits (CG000086_REV C).
Gilchuk P., Bombardi R.G., Erasmus J.H., Tan Q., Nargi R., Soto C., Abbink P., Suscovich T.J., Durnell L.A., Khandhar A., Archer J., Liang J., Fouch M.E., Davidson E., Doranz B.J., Jones T., Larson E., Ertel S., Granger B., Fuerte-Stone J., Roy V., Broge T., Linnekin T.C., Linde C.H., Gorman M.J., Nkolola J., Alter G., Reed S.G., Barouch D.H., Diamond M.S., Crowe JE J.r., Van Hoeven N., Thackray L, & Carnahan R. (2020). Integrated pipeline for the accelerated discovery of antiviral antibody therapeutics. Nature biomedical engineering, 4(11), 1030-1043.
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10

Single Nuclei RNA-seq of Mouse Ovaries

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The ovaries were collected from the mice and immediately froze in liquid nitrogen. Before the Single nuclei RNA‐seq (sNuc‐seq) started, the ovaries were minced and homogenized in a nuclei extraction buffer (Tris 10 mM, Tween‐20 0.03%, NaCl 146 mM, CaCl2 1 mM, MgCl2 21 mM, and BSA 0.01%). The nuclei were immediately loaded on the 10× Chromium controller (10× Genomics) with Single Cell 3′ v3.1 chemistry according to the manufacturer's protocol after FACS purification. For each sample, ~ 10,000 nuclei were loaded in one channel of a Chromium Chip (10× Genomics). The cDNA generation and library preparation were performed according to the manufacturer's protocol and sequenced using the Illumina HiSeq platform. The data was initially processed by Cell Ranger and analyzed by Seurat v4.0.
Guo X., Zhu Y., Guo L., Qi Y., Liu X., Wang J., Zhang J., Cui L., Shi Y., Wang Q., Liu C., Lu G., Liu Y., Li T., Hong S., Qin Y., Xiong X., Wu H., Huang L., Huang H., Gu C., Li B, & Li J. (2023). BCAA insufficiency leads to premature ovarian insufficiency via ceramide‐induced elevation of ROS. EMBO Molecular Medicine, 15(4), e17450.
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