Chromium single cell 3 kit

Manufactured by 10x Genomics

Sourced in United States

The Chromium Single Cell 3' kit is a lab equipment product developed by 10x Genomics. The kit enables the analysis of gene expression at the single-cell level.

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Lab products found in correlation

Hiseq 2500, by Illumina (4 mentions) Novaseq 6000, by Illumina (3 mentions) Chromium controller, by 10x Genomics (2 mentions) Chromium single cell instrument, by 10x Genomics (2 mentions) Tapestation, by Agilent Technologies (2 mentions) Nextseq 500, by Illumina (2 mentions) Bioanalyzer hs dna kit, by Agilent Technologies (2 mentions) Easysep human b cell isolation kit, by STEMCELL (1 mentions) Hiseq 3000, by Illumina (1 mentions) 2100 bioanalyzer, by Illumina (1 mentions) Nextseq 550 system, by Illumina (1 mentions) Novaseq 6000 sequencer, by Illumina (1 mentions) Bioanalyzer, by Agilent Technologies (1 mentions) Dna hs kit, by Agilent Technologies (1 mentions) Qubit fluorimeter, by Thermo Fisher Scientific (1 mentions) Nextseq 550, by Illumina (1 mentions) Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions) Hiseq 2500 4000, by Illumina (1 mentions) Kapa library quantification, by Roche (1 mentions) Bioanalyzer high sensitivity dna kit, by Agilent Technologies (1 mentions)

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17 protocols using chromium single cell 3 kit

Single-cell RNA-seq from mouse brain

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Single-nuclei separation, barcoding, and cDNA generation were performed following the manufacturer’s instruction using the Chromium single cell 3′ kit (V3, 10X Genomics PN-1000073). cDNA concentration and quality were measured using Qubit Fluorimeter (Thermo Fisher) and Tape Station (Agilent), respectively, and was stored at –20°C until library preparation.
Libraries were generated from all samples at the same time (eight total samples, 2 WT/2 KO Vglut2 cKO model; 2 WT/2KO IP3R2 KO model) following the manufacturer’s instructions using the Chromium single cell 3′ kit (V3, 10X Genomics PN-1000075). Library quality was assessed with a Tape Station (Agilent). NovaSeq sequencing was performed at the UCSF Center for Advanced Technology, at ~300 million reads/sample (60,000 reads/cell).

Farhy-Tselnicker I., Boisvert M.M., Liu H., Dowling C., Erikson G.A., Blanco-Suarez E., Farhy C., Shokhirev M.N., Ecker J.R, & Allen N.J. (2021). Activity-dependent modulation of synapse-regulating genes in astrocytes. eLife, 10, e70514.

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Single-cell RNA-seq of Isolated B Cells

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B cells were isolated from frozen-thawed PBMCs using the EasySep Human B cell Isolation Kit (STEMCELL Technologies) on a RoboSep platform. Cells were encapsulated in droplets using the Chromium Controller (10x Genomics). Reverse transcription and cDNA amplification were performed using the Chromium Single Cell 3′ Kit (10x Genomics). All steps were done according to the manufacturer’s instructions. One hundred nanograms of cDNA (25% of generated cDNA) was used as input for library preparation. Libraries were sequenced using the Illumina HiSeq 3000 platform eight lanes of sequencing at 26 bp–8 bp–98 bp according to 10x Genomics sequencing recommendations.

Holla P., Dizon B., Ambegaonkar A.A., Rogel N., Goldschmidt E., Boddapati A.K., Sohn H., Sturdevant D., Austin J.W., Kardava L., Yuesheng L., Liu P., Moir S., Pierce S.K, & Madi A. (2021). Shared transcriptional profiles of atypical B cells suggest common drivers of expansion and function in malaria, HIV, and autoimmunity. Science Advances, 7(22), eabg8384.

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Single-Cell RNA-Seq Library Preparation

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The suspensions of single cells were loaded onto Chromium microfluidic chips to generate single‐cell gel bead emulsions using the Chromium Controller from 10X Genomics, following the manufacturer's instructions. The mRNA from the samples was processed with the Chromium Single‐Cell 3′ kit from 10X Genomics, and full‐length, barcoded cDNA was generated by PCR amplification to produce enough material for library construction. The library were assessed using an Agilent Bioanalyzer 2100, and sequencing was carried out on an Illumina NovaSeq 6000 (LC‐Bio Technology Co.Ltd., Hangzhou, China) using paired‐end multiplexing with a read length of 150 bp.⁷¹, ⁷²

Ji K., Chen L., Wang X., Wen B., Yang F., Deng W., Chen Y., Zhang G, & Liu H. (2023). Integrating single‐cell RNA sequencing with spatial transcriptomics reveals an immune landscape of human myometrium during labour. Clinical and Translational Medicine, 13(4), e1234.

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Single-cell RNA-seq Library Preparation

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The same method was used to make the single-cell suspension as described above. We first collected GFP- and Hoechst-positive single cells in a 1.5-ml Eppendorf tube with 0.3 ml of collection buffer [phosphate-buffered saline (PBS) + 0.04% bovine serum albumin]. The cells were spun down on a centrifuge by 700g for 10 min. We used the Chromium Single Cell 3′ Kit (v3) of 10X Genomics. The libraries were prepared according to the standard user guide (CG000315 Rev. B) from 10X without any modifications.

Ma D., Herndon N., Le J.Q., Abruzzi K.C., Zinn K, & Rosbash M. (2023). Neural connectivity molecules best identify the heterogeneous clock and dopaminergic cell types in the Drosophila adult brain. Science Advances, 9(8), eade8500.

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Evaluating Nuclei Suspension Quality for scRNA-Seq

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Timing: 30 min

Here we describe how we evaluate the quality of the nuclei suspension and calculate the nuclei concentration. In terms of quality control, we consider this protocol successful if 1) nuclei appear as a single-nuclei solution and are not clumped together, and 2) nuclei look intact. For these quality control steps the following staining approaches were used.

13.

Assess nuclei integrity with Ethidium Homodimer-1 (EthD-1) staining.a.

Stain 10 μL of nuclei suspension (from 11e) with 4 μM EthD-1 (Figure 1B). EthD-1 emits red fluorescence upon binding to DNA and will show DNA blebbing if the sample quality is low. Visualize stained nuclei under an epifluorescence microscope in the red channel.

14.

Count nuclei with Trypan Blue staininga.

Stain 10 μL of nuclei suspension (from Step 11e) with 10 μL of premade 0.4% Trypan Blue.

Load the stained sample on a hemocytometer and visualize the nuclei under a bright field microscope. Count blue/purple stained nuclei.

15.

Adjust the final concentration of nuclei to 1000 nuclei/μL with NSB.

16.

Continue with preparation of droplet-based single-nuclei RNA-seq libraries using the Chromium Single Cell 3′ kit (10× Genomics) according to the manufacturer’s protocol

Ayhan F., Douglas C., Lega B.C, & Konopka G. (2021). Nuclei isolation from surgically resected human hippocampus. STAR Protocols, 2(4), 100844.

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Single-cell RNA-sequencing of cells

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We used the Chromium Single Cell 3′ kit (v3) of 10x Genomics for performing scRNA-sequencing. The single cell suspension prepared above was loaded onto a Chromium Single-Cell instrument (10x Genomics) to generate single-cell barcoded droplets (GEMs) as detailed in manufacture’s protocol. The resulting libraries were pooled and sequenced across two lanes on an Illumina HiSeq2500 instrument in High-output mode by Novogene. Reads were aligned and subsequent analyses performed using the Cell Ranger (Pipeline). We obtained 68k reads per cell with a median genes per cell of 1,017 and median UMI count per cell of 2,040. The accession number for the scRNA-seq data is GEO database: GSE172059.

Gao F., Li C., Danopoulos S., Al Alam D., Peinado N., Webster S., Borok Z., Kohbodi G.A., Bellusci S, & Minoo P. (2022). Hedgehog-responsive PDGFRa(+) fibroblasts maintain a unique pool of alveolar epithelial progenitor cells during alveologenesis. Cell reports, 39(1), 110608.

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Single-cell RNA-sequencing of cells

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Chromium Single Cell 3' Kit (v3.1) for SARGENT

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We used the Chromium Single Cell 3’ Kit (v3.1) from 10× Genomics for SARGENT. We followed the manufacturer’s instructions for preparing single-cell suspensions. We used a cell counter to measure the number of cells and viability and used cell preparations with greater than 95% cell viability.

Hong C.K., Ramu A., Zhao S, & Cohen B.A. (2024). Effect of genomic and cellular environments on gene expression noise. Genome Biology, 25, 137.

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Single-cell RNA-seq of Mouse Samples

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Female samples were processed at the Scripps Genomics core (La Jolla, CA, USA). Male samples were prepared on a different day by the Next Generation Sequencing (NGS) Core at the Salk institute (La Jolla, CA, USA) in similar conditions. For each sample (n = 3 mice), 15,000 cells were processed for scRNAseq using the Chromium Single Cell 3′ kit (v3.1 Chemistry) from 10× Genomics (Pleasanton, CA, USA) according to the manufacturer’s protocols. The cDNA was amplified by 11 cycles. Analysis of the cDNA and libraries was conducted using Bioanalyzer or TapeStation (Agilent, Santa Clara, CA, USA). The libraries were sequenced multiple times by Nextseq 550 systems and Novaseq 6000 sequencer (Illumina, San Diego, CA, USA) to reach the targeted minimal sequencing depth (20,000 reads/cell).

Delcroix V., Mauduit O., Lee H.S., Ivanova A., Umazume T., Knox S.M., de Paiva C.S., Dartt D.A, & Makarenkova H.P. (2023). The First Transcriptomic Atlas of the Adult Lacrimal Gland Reveals Epithelial Complexity and Identifies Novel Progenitor Cells in Mice. Cells, 12(10), 1435.

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Single-Cell RNA-Seq of PDOs

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Irradiated (5 Gy/day over 5 days) and non-irradiated PDOs were dissociated into single cells after a 6-day recovery period. Libraries were prepared aiming for 7000 cell recovery, using 11 cycles of amplification of cDNA and 12 cycles for indexing PCR using the Chromium single cell 3’ kit (10X Genomics, USA). Sequencing was performed using a NextSeq™ 550 HIGH 150 flow cell and NGS platform.

Wanigasooriya K., Barros-Silva J.D., Tee L., El-asrag M.E., Stodolna A., Pickles O.J., Stockton J., Bryer C., Hoare R., Whalley C.M., Tyler R., Sillo T., Yau C., Ismail T, & Beggs A.D. (2022). Patient Derived Organoids Confirm That PI3K/AKT Signalling Is an Escape Pathway for Radioresistance and a Target for Therapy in Rectal Cancer. Frontiers in Oncology, 12, 920444.

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