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Genomics chromium instrument

Manufactured by 10x Genomics
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Sourced in United States

The 10x Genomics Chromium instrument is a microfluidics-based platform designed for single-cell analysis and high-throughput genomics applications. The core function of the Chromium instrument is to partition individual cells or nuclei into nanoliter-scale droplets, enabling the simultaneous processing of thousands of samples in a single experiment.

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Lab products found in correlation

Bioanalyzer high sensitivity dna kit, by Agilent Technologies (5 mentions) Chromium single cell 5 library gel bead kit, by 10x Genomics (3 mentions) Novaseq s4 platform, by Illumina (2 mentions) Totalseq antibody, by BioLegend (1 mentions) Chromium single cell 5 library gel bead kit v2, by 10x Genomics (1 mentions) Cell ranger pipeline, by 10x Genomics (1 mentions) Agilent 2200 tapestation system, by Agilent Technologies (1 mentions) Novaseq platform, by Illumina (1 mentions) Novaseq s4 300 cycle platform, by Illumina (1 mentions)

8 protocols using genomics chromium instrument

1

Single-Cell Transcriptomics with Multiplexing

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Cell hashing was used to combine multiple samples into the same single-cell emulsion channel41 (link). The mouse cells obtained from different stimulation conditions were stained with TotalSeq antibodies (BioLegend anti-mouse hashtags 1–8; used at 1:100 dilution), washed 5 times at 4 °C and pooled in PBS with 0.04% BSA according to the manufacturer’s protocol. Next, 55,000 cells were loaded onto a 10x Genomics Chromium instrument (10x Genomics) according to the manufacturer’s instructions. The scRNA-seq libraries were processed using a Chromium Single Cell 3′ Library & Gel Bead v3 kit (10x Genomics) with modifications for generating hashtag libraries41 (link). Quality control for amplified cDNA libraries and final sequencing libraries was performed using a Bioanalyzer High Sensitivity DNA kit (Agilent). scRNA-seq and hashing libraries were normalized to 4 nM concentration and pooled. The pooled libraries were paired-end sequenced on a NovaSeq S4 platform targeting an average sequencing depth of 20,000 reads per cell for gene expression libraries, and on a NovaSeq S4 or SP platform targeting 5,000 reads per cell for hashtag libraries.
Cui A., Huang T., Li S., Ma A., Pérez J.L., Sander C., Keskin D.B., Wu C.J., Fraenkel E, & Hacohen N. (2023). Dictionary of immune responses to cytokines at single-cell resolution. Nature, 625(7994), 377-384.
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2

Single-Cell RNA-Seq of Chronic Lymphocytic Leukemia

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The sample processed for scRNA-seq was obtained from the CLL Research Consortium from a patient consented to an IRB approved research protocol. Cells were thawed and washed twice in RPMI and 10% FCS before undergoing dead cell depletion (Miltenyi Biotec; 130-090-101). CLL/B cells were purified using CD19 negative selection kit (Biolegend; 480082) and CD19 negative immune cells were purified using CD19 positive magnetic beads (Biolegend; 480106) and viable cells mixed before being washed in PBS with 0.04% BSA twice and re-suspended in PBS with 0.04% BSA (Life Technologies; AM2616) at the cell concentration of 1000 cells/μL. 17,000 cells were loaded onto a 10x Genomics Chromium instrument (10x Genomics) according to the manufacturer’s instructions. The single cell RNA-seq libraries were processed using Chromium single cell 5’ library & gel bead kit (10x Genomics). Quality controls for amplified cDNA libraries and final sequencing libraries were performed using Bioanalyzer High Sensitivity DNA Kit (Agilent). The libraries were normalized to 4 nM concentration and pooled in a volume ratio of 4:1. The pooled libraries were sequenced on Illumina NovaSeq S2 100 cycle platform. The sequencing parameters were: Read 1 of 28 bp, Read 2 of 91 bp and Index 1 of 8 bp.
Lazarian G., Yin S., Hacken E.T., Sewastianik T., Uduman M., Font-Tello A., Gohil S.H., Li S., Kim E., Joyal H., Billington L., Witten E., Zheng M., Huang T., Severgnini M., Lefebvre V., Rassenti L.Z., Gutierrez C., Georgopoulos K., Ott C.J., Wang L., Kipps T.J., Burger J.A., Livak K.J., Neuberg D.S., Baran-Marszak F., Cymbalista F., Carrasco R.D, & Wu C.J. (2021). A hotspot mutation in transcription factor IKZF3 drives B cell neoplasia via transcriptional dysregulation. Cancer cell, 39(3), 380-393.e8.
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3

Single-cell RNA and TCR Sequencing

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Viable cells were resuspended in PBS with 0.04% BSA at a cell concentration of 1000 cells/μL. 40,000 cells were loaded onto a 10x Genomics Chromium instrument (10x Genomics) according to the manufacturer’s instructions. The scRNA-seq libraries were processed using Chromium single cell 5’ library & gel bead kit v2 and coupled scTCR-seq libraries were obtained using Chromium single cell V(D)J enrichment kit (human T cell) (10x Genomics). Quality control for amplified cDNA libraries and final sequencing libraries were performed using Bioanalyzer High Sensitivity DNA Kit (Agilent). Both scRNA-seq and scTCR-seq libraries were normalized to 4nM concentration and pooled in a volume ratio of 4:1. The pooled libraries were sequenced on Illumina NovaSeq S4 platform. The sequencing parameters were: Read 1 of 26bp, Read 2 of 90bp, index 1 of 10bp and index 2 of 10bp.
Haradhvala N.J., Leick M.B., Maurer K., Gohil S.H., Larson R.C., Yao N., Gallagher K.M., Katsis K., Frigault M.J., Southard J., Li S., Kann M.C., Silva H., Jan M., Rhrissorrakrai K., Utro F., Levovitz C., Jacobs R.A., Slowik K., Danysh B.P., Livak K.J., Parida L., Ferry J., Jacobson C., Wu C.J., Getz G, & Maus M.V. (2022). Distinct cellular dynamics associated with response to CAR-T therapy for refractory B-cell lymphoma. Nature medicine, 28(9), 1848-1859.
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4

Single-cell RNA and TCR sequencing

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Viable cells were resuspended in PBS with 0.04% BSA at a cell concentration of 1000 cells/μL. 17,000 cells were loaded onto a 10x Genomics Chromium™ instrument (10x Genomics) according to the manufacturer’s instructions. The scRNA-seq libraries were processed using Chromium™ single cell 5’ library & gel bead kit and coupled scTCR-seq libraries were obtained using Chromium™ single cell V(D)J enrichment kit (human T cell) (10x Genomics). Quality control for amplified cDNA libraries and final sequencing libraries were performed using Bioanalyzer High Sensitivity DNA Kit (Agilent). Both scRNA-seq and scTCR-seq libraries were normalized to 4nM concentration and pooled in a volume ratio of 4:1. The pooled libraries were sequenced on Illumina NovaSeq S4 platform. The sequencing parameters were: Read 1 of 150bp, Read 2 of 150bp and Index 1 of 8bp. The sequencing data were processed using the Cell Ranger pipeline (version 2.1.0 using hg19 reference, 10x Genomics), including sequencing data demultiplexing, data alignment and TCR clonotype assembly.
Braun D.A., Street K., Burke K.P., Cookmeyer D.L., Denize T., Pedersen C.B., Gohil S.H., Schindler N., Pomerance L., Hirsch L., Bakouny Z., Hou Y., Forman J., Huang T., Li S., Cui A., Keskin D.B., Steinharter J., Bouchard G., Sun M., Pimenta E.M., Xu W., Mahoney K.M., McGregor B.A., Hirsch M.S., Chang S.L., Livak K.J., McDermott D.F., Shukla S.A., Olsen L.R., Signoretti S., Sharpe A.H., Irizarry R.A., Choueiri T.K, & Wu C.J. (2021). Progressive immune dysfunction with advancing disease stage in renal cell carcinoma. Cancer cell, 39(5), 632-648.e8.
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5

Single-Cell Sequencing Library Preparation

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Seven thousand cells were loaded onto a 10x Genomics Chromium instrument (10x Genomics, Pleasanton, CA, USA) to create single-cell Gel Bead-in-Emulsions (GEMs). cDNA libraries were constructed using 10x Chromium Single cell 3’ Reagent Kits v3.1, following the manufacturer’s instructions. Briefly, amplified cDNA products were cleaned using the SPRI Select Reagent Kit (10x Genomics). Indexed sequencing libraries were constructed and barcoded sequencing libraries were quantified using the Qubit 2.0 ds HS Assay Kit (Invitrogen). The quality of the libraries was checked using an Agilent 2200 Tapestation System (Agilent Technologies,Inc. Santa Clara, CA). Finally, libraries were sequenced using the Illumina NovaSeq platform.
Nakamura Y., Matsumoto H., Wu C.H., Fukaya D., Uni R., Hirakawa Y., Katagiri M., Yamada S., Ko T., Nomura S., Wada Y., Komuro I., Nangaku M., Inagi R, & Inoue T. (2023). Alpha 7 nicotinic acetylcholine receptors signaling boosts cell-cell interactions in macrophages effecting anti-inflammatory and organ protection. Communications Biology, 6, 666.
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6

Identifying Regulatory T Cells in Injured Lymph Nodes

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For single cell RNAseq (scRNAseq) and single cell TCRseq (scTCRseq), viable lymph node FoxP3 GFP+ cells were sorted from injured or uninjured Foxp3DTR mice at 7 days after injury. The sorted FoxP3 GFP+ cells were washed and resuspended in sorting buffer at a cell concentration of 1000 cells/µL. About 17,000 mouse cells were loaded onto a 10x Genomics Chromium™ instrument (10x Genomics) according to the manufacturer’s recommendations. The scRNAseq libraries were processed using Chromium™ single cell 5’ library & gel bead kit (10x Genomics). Matched scTCRseq libraries were prepared using 2 µL of post cDNA amplification material and Chromium Single Cell V(D)J Enrichment Kit, Mouse T Cell. Quality controls for amplified cDNA libraries and final sequencing libraries were performed using Bioanalyzer High Sensitivity DNA Kit (Agilent). The sequencing libraries for scRNAseq and scTCRseq were normalized to 4nM concentration and pooled using a volume ratio of 4:1. The pooled sequencing libraries were sequenced on Illumina NovaSeq S4 300 cycle platform. The sequencing parameters were: Read 1 of 150bp, Read 2 of 150bp and Index 1 of 8bp. The sequencing data were demultiplexed and aligned to GRCm38 using cell ranger version 3.1.0 pipeline (10x Genomics).
Guo F., Hancock B., Griffith A., Lin H., Howard K., Keegan J., Zhang F., Chicoine A., Cahill L., Ng J, & Lederer J. (2022). Distinct Injury Responsive Regulatory T Cells Identified by Multi-Dimensional Phenotyping. Frontiers in Immunology, 13, 833100.
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7

Single-cell gene expression profiling

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The sorted cells were then loaded onto a 10× Genomics Chromium
instrument (10× Genomics, Pleasanton, USA), generating single-cell gel
beads in emulsion (GEMs), 200,000 live CD45+ cells per experiment,
and processed as described previously to sequence the 3’ end of
transcripts [25 (link)].
Weinstock A., Brown E.J., Garabedian M.L., Pena S., Sharma M., Lafaille J., Moore K.J, & Fisher E.A. (2019). Single-Cell RNA Sequencing of Visceral Adipose Tissue Leukocytes Reveals that Caloric Restriction Following Obesity Promotes the Accumulation of a Distinct Macrophage Population with Features of Phagocytic Cells. Immunometabolism, 1, e190008.
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8

Single-cell gene expression profiling

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The sorted cells were then loaded onto a 10× Genomics Chromium
instrument (10× Genomics, Pleasanton, USA), generating single-cell gel
beads in emulsion (GEMs), 200,000 live CD45+ cells per experiment,
and processed as described previously to sequence the 3’ end of
transcripts [25 (link)].
Weinstock A., Brown E.J., Garabedian M.L., Pena S., Sharma M., Lafaille J., Moore K.J, & Fisher E.A. (2019). Single-Cell RNA Sequencing of Visceral Adipose Tissue Leukocytes Reveals that Caloric Restriction Following Obesity Promotes the Accumulation of a Distinct Macrophage Population with Features of Phagocytic Cells. Immunometabolism, 1, e190008.
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