Chromium single cell 3 reagent kit v3

Manufactured by 10x Genomics

Sourced in United States

The Chromium Single Cell 3' Reagent Kit v3 is a laboratory equipment product offered by 10x Genomics. The kit is designed to enable single-cell RNA sequencing analysis. It provides the necessary reagents and materials to prepare and process single-cell samples for downstream genomic analysis.

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Spelling variants of this product

Chromium Single Cell 3′ v3 (8 citations) Chromium Single Cell 3′ Reagents Kits v3.1 (3 citations) Reagent Kit v3.1 (2 citations)

97 protocols using chromium single cell 3 reagent kit v3

Single-cell RNA sequencing of iPSCs, neurons

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For iPSC, iNeurons, and iLowerMotorneurons, single-cell RNA sequencing was performed using Chromium Single Cell 3’ Reagent kit V3.1 (PN-1000128) and 2.5×10⁴ cells per condition were loaded into the 10x Genomics chip G. For cortical and hypothalamic neurons, single-cell suspensions were processed by the Chromium Controller (10x Genomics) using Chromium Single Cell 3’ Reagent Kit v3 (PN-1000075) according to the manufacturer’s specifications. On average, 15,000 cells from each 10x reaction were directly loaded into one inlet of the 10x Genomics chip. Barcoded libraries were sequenced using the Illumina Novaseq 6000 (one lane per 10x chip position) with 75 bp paired-end reads to an average depth of approximately 5×10⁴ reads per cell.

Pantazis C.B., Yang A., Lara E., McDonough J.A., Blauwendraat C., Peng L., Oguro H., Kanaujiya J., Zou J., Sebesta D., Pratt G., Cross E., Blockwick J., Buxton P., Kinner-Bibeau L., Medura C., Tompkins C., Hughes S., Santiana M., Faghri F., Nalls M.A., Vitale D., Ballard S., Qi Y.A., Ramos D.M., Anderson K.M., Stadler J., Narayan P., Papademetriou J., Reilly L., Nelson M.P., Aggarwal S., Rosen L.U., Kirwan P., Pisupati V., Coon S.L., Scholz S.W., Priebe T., Öttl M., Dong J., Meijer M., Janssen L.J., Lourenco V.S., van der Kant R., Crusius D., Paquet D., Raulin A.C., Bu G., Held A., Wainger B.J., Gabriele R.M., Casey J.M., Wray S., Abu-Bonsrah D., Parish C.L., Beccari M.S., Cleveland D.W., Li E., Rose I.V., Kampmann M., Aristoy C.C., Verstreken P., Heinrich L., Chen M.Y., Schüle B., Dou D., Holzbaur E.L., Zanellati M.C., Basundra R., Deshmukh M., Cohen S., Khanna R., Raman M., Nevin Z.S., Matia M., Van Lent J., Timmerman V., Conklin B.R., Chase K.J., Zhang K., Funes S., Bosco D.A., Erlebach L., Welzer M., Kronenberg-Versteeg D., Lyu G., Arenas E., Coccia E., Sarrafha L., Ahfeldt T., Marioni J.C., Skarnes W.C., Cookson M.R., Ward M.E, & Merkle F.T. (2022). A reference human induced pluripotent stem cell line for large-scale collaborative studies. Cell stem cell, 29(12), 1685-1702.e22.

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Single-cell RNA-seq of COVID-19 lung samples

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scRNA-seq was done according to the manufacturer instructions (10X genomics). Briefly, after quickly thawing the frozen lung single-cell suspension in a water bath, 2X10⁶ cells were taken for downstream processing. Cell suspensions were enriched for CD45 by using NHP CD45-microbeads according to the manufacturer’s instruction (Milteny Biotec). After then, CD45⁺ enriched lung single cell suspensions were subjected to droplet-based massively parallel scRNA-seq using Chromium Single Cell 3’ (v3) Reagent Kit in the BSL-3 laboratory as per manufacturer’s instructions (10x Genomics). Briefly, cell suspensions were loaded at 1,000 cells/μl to capture 10,000 cells/lane. The 10x Chromium Controller generated GEM droplets, where each cell was labeled with a specific barcode, and each transcript was labeled with a unique molecular identifier (UMI) during reverse transcription. The barcoded cDNA was isolated and removed from the BSL-3 space for library generation. The cDNA underwent 11 cycles of amplification, followed by fragmentation, end repair, A-tailing, adapter ligation, and sample index PCR as per the manufacturer’s instructions. Libraries were sequenced on a NovaSeq S4 (200 cycles) flow cell, targeting 50,000 read pairs/cell.

Esaulova E., Das S., Singh D.K., Choreño-Parra J.A., Swain A., Arthur L., Rangel-Moreno J., Ahmed M., Singh B., Gupta A., Fernández-López L.A., Garcia-Hernandez M.D., Bucsan A., Moodley C., Mehra S., García-Latorre E., Zuniga J., Atkinson J., Kaushal D., Artyomov M.N, & Khader S.A. (2020). The immune landscape in tuberculosis reveals populations linked to disease and latency. Cell host & microbe, 29(2), 165-178.e8.

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Single-Cell RNA-Seq of FACS-Purified CD45+ Cells

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Viable CD45⁺ cells from each subject purified by FACS were then resuspended at a concentration of 1,000 cells/μl, following the instructions of single-cell 3′ solution v3 reagent kit (10X Genomics) to capture around 10,000 cells per sample. The protoplast suspension was then loaded into Chromium microfluidic chips and barcoded with a 10X Chromium Controller (10X Genomics). RNA from the barcoded cells was subsequently reverse-transcribed, and sequencing libraries were constructed with reagents from a Chromium Single Cell 3′ v3 Reagent Kit (10X Genomics) according to the manufacturer’s instructions. Sequencing was performed with Illumina (NovaSeq 6000) according to the manufacturer’s instructions (Illumina). Each sample was sequenced for at least 150 Gb.

Xie J., Gu A., He H., Zhao Q., Yu Y., Chen J., Cheng Z., Zhou P., Zhou Q, & Jin M. (2023). Autoimmune thyroid disease disrupts immune homeostasis in the endometrium of unexplained infertility women—a single-cell RNA transcriptome study during the implantation window. Frontiers in Endocrinology, 14, 1185147.

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Single-Cell RNA-Seq Protocol for 10x Genomics

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Each qualified sample (

greater than 80 percent

viable cells) that consisted of

approximately 10,000

cells was used to build cDNA libraries for the scRNA-seq data based on the manufacturer’s protocol with Chromium Single Cell 3′ v3 Reagent Kit (PN-1000075; 10x Genomics). The workflow of building cDNA libraries was as follows. After preparing the Master Mix on ice, qualified cell suspensions were added to the Master Mix and

75 microliters

of the mixtures were loaded into each of the wells in row 1 of the 10x Chip Wells. Forty microliters of Single Cell 3′ v3 Gel Beads were dispensed into each of the wells in row 2. Partitioning Oil was added in row 3 in two

140 microliter

aliquots, followed by running the Chromium Single Cell B program in the Chromium Controller. Next, reverse transcription incubation was performed according to the following protocol: 53°C for 45 min, 85°C for 5 min, and 4°C hold. Nine libraries were sequenced on NovaSeq6000 (Illumina) separately at the sequencing depth of 500 million reads per cell. Detailed information about scRNA-seq cDNA libraries is provided in Table S1.

Zhang R., Chen S., Wang Z., Ye L., Jiang Y., Li M., Jiang X., Peng H., Guo Z., Chen L., Zhang R., Niu Y., Aschner M., Li D, & Chen W. (2023). Assessing the Effects of Nicotinamide Mononucleotide Supplementation on Pulmonary Inflammation in Male Mice Subchronically Exposed to Ambient Particulate Matter. Environmental Health Perspectives, 131(7), 077006.

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Single-cell RNA-seq with 10x Genomics

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Cells were loaded for an expected recovery of 10,000 cells per channel. The chip loaded with single cell suspension was placed on a 10x Genomics Chromium Controller Instrument (10x Genomics, Pleasanton, CA, USA) to generate single cell droplets containing uniquely barcoded GEMs (Gel Bead-In EMulsions). Single-cell RNA-seq libraries were obtained following the 10x Genomics recommended protocol, using the reagents included in the Chromium Single Cell 3′ v3 Reagent Kit. The libraries were sequenced on the NovaSeq S2 flow cell (Illumina, 100 cycles). Sample demultiplexing, alignment, filtering, and UMI counting were obtained by using Cell Ranger 3.0.1.

Shamsi F., Piper M., Ho L.L., Huang T.L., Gupta A., Streets A., Lynes M.D, & Tseng Y.H. (2021). Vascular smooth muscle-derived TRPV1-positive progenitors are a source of cold-induced thermogenic adipocytes. Nature metabolism, 3(4), 485-495.

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Single-cell RNA sequencing using 10X Chromium

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The cell suspension was loaded into Chromium microfluidic chips with 3’ v3 chemistry and barcoded with a 10× Chromium Controller (10× Genomics). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries constructed with reagents from a Chromium Single Cell 3’ v3 reagent kit (10× Genomics) according to the manufacturer’s instructions (https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstrated-protocol-single-cell-protocols-cell-preparation-guide). Sequencing was performed with Illumina (HiSeq 2000) according to the manufacturer’s instructions Illumina.

Wu B., Yuan Y., Liu J., Shang H., Dong J., Liang X., Wang D., Chen Y., Wang C., Zhou Y., Jing H, & Cheng W. (2021). Single-cell RNA sequencing reveals the mechanism of sonodynamic therapy combined with a RAS inhibitor in the setting of hepatocellular carcinoma. Journal of Nanobiotechnology, 19, 177.

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Single-cell RNA-seq of Mouse Aorta

Cited 1 time

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Mouse infrarenal abdominal aortas were collected and digested in an enzymatic mix (DMEM containing 10 mg/ml collagenase type II (C6885, Sigma Aldrich) and 1 mg/ml elastase (LS002292, Worthington Biochemistry)) for 15 minutes at 37 °C^{8 (link)}. The tissue suspension was filtered with a 40 μm cell strainer, then centrifuged at 500 g for 5 min. Cells were resuspended with DMEM containing 10% FBS. Single-cell suspensions from 4 aortas per group were pooled together as one sample. 5000 cells per sample were loaded on a Chromium Controller (10x Genomics). The scRNA-seq libraries were constructed using the Chromium Single Cell 3’ v3 Reagent Kit according to the manufacturers guidelines (10x Genomics). cDNA libraries were uniquely sample indexed and pooled for sequencing. A MiSeq (Illumina) sequencing run was used to sample balance on a NovaSeq S1 flowcell (Illumina) using a 28×91bp sequencing reaction targeting >50,000 reads/cell.

Yang H., Zhou T., Stranz A., DeRoo E, & Liu B. (2021). Single-cell RNA sequencing reveals heterogeneity of vascular cells in early stage murine abdominal aortic aneurysm. Arteriosclerosis, thrombosis, and vascular biology, 41(3), 1158-1166.

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Single-Cell RNA-seq and HTO Profiling

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Libraries were prepared using the Chromium Single Cell 3′ v3 reagent kit (10x Genomics, 1000075) following the 10x Genomics User Guide (CG000183 Rev A), with the only modification being cell overloading. All libraries were sequenced on an Illumina NovaSeq S4 flowcell. Target read counts were 30,000 reads per cell for RNA libraries and 2,000 reads per cell for HTO libraries.

Swanson E., Reading J., Graybuck L.T, & Skene P.J. (2022). BarWare: efficient software tools for barcoded single-cell genomics. BMC Bioinformatics, 23, 106.

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Multimodal Single-Cell Profiling

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Single-cell suspensions were dispensed separately onto the Chromium Controller (10x Genomics, Pleasanton, CA) with a target of 10,000 cells/sample. The scRNA-seq library was constructed by using the Chromium Single-Cell 3′ v3 Reagent Kit (10x Genomics). Cells were mixed with barcoded primer-linked gel beads. Each cell was barcoded with a unique index, and every transcript within a single cell was barcoded with a unique molecular identifier (UMI). cDNAs were pooled, truncated, and amplified to generate cDNA libraries that were sequenced using a Next Generation sequencer NovaSeq 6000 (Illumina, Inc., San Diego, CA) in a pair-end fashion to obtain more than 80,000 reads per cell.
Single-nuclei suspensions were prepared according to the manufacturer’s instructions (10x Genomics). Nuclei were loaded with a capture target of 10,000 nuclei per sample. ScATAC-seq libraries were prepared for sequencing according to the 10x Genomics scATAC-seq solution protocol. ScATAC-seq libraries were sequenced by using PE150 sequencing on an Illumina NovaSeq with a target depth of 25,000 reads per nucleus.
Processing and analyses of scRNA-seq and scATAC-seq data are presented in the Materials and Methods of the Supplemental Material.

Zhang C., Li Y., Chakraborty A., Li Y., Rebello K.R., Ren P., Luo W., Zhang L., Lu H.S., Cassis L.A., Coselli J.S., Daugherty A., LeMaire S.A, & Shen Y.H. (2022). Aortic Stress Activates an Adaptive Program in Thoracic Aortic Smooth Muscle Cells That Maintains Aortic Strength and Protects Against Aneurysm and Dissection in Mice. Arteriosclerosis, thrombosis, and vascular biology, 43(2), 234-252.

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Analyzing Skin Immune Responses to S. aureus

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At 2 days (48 hours) and 5 days (128 hours) after intradermal injection of S. aureus, single-cell suspensions were obtained from skin as described above. Cells were treated with anti-CD16/32 to neutralize Fc receptors. Cells then were stained with anti-CD45.2. and 7-AAD, and live leukocytes were isolated using a FACS Aria II cell sorter (BD Biosciences). Single-cell RNA sequencing was performed using a Chromium Single Cell 3’ V3 Reagent kit (10X Genomics, Pleasanton, CA, USA) according to the manufacturer’s protocol. For additional information on single-cell RNA sequencing and its analysis methods, please see the Supplementary Data.

Park Y.J., Kang B.H., Kim H.J., Oh J.E, & Lee H.K. (2022). A Microbiota-Dependent Subset of Skin Macrophages Protects Against Cutaneous Bacterial Infection. Frontiers in Immunology, 13, 799598.

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