Chromium single cell 3 library kit v2

Manufactured by 10x Genomics

Sourced in United States

The Chromium Single Cell 3' library kit v2 is a laboratory equipment product manufactured by 10x Genomics. The core function of this kit is to enable the generation of single-cell gene expression libraries for next-generation sequencing.

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Lab products found in correlation

Chromium controller, by 10x Genomics (5 mentions) Chromium single cell 3 reagent kits v2, by 10x Genomics (4 mentions) Lightcycler 96 system, by Roche (4 mentions) Dynabeads myone silane beads, by Thermo Fisher Scientific (4 mentions) Spriselect beads, by Beckman Coulter (4 mentions) Hiseq 4000, by Illumina (2 mentions) Moflo xdp, by Beckman Coulter (2 mentions) Novaseq 6000, by Illumina (1 mentions) Nextseq 500, by Illumina (1 mentions) Single cell 3 chip, by 10x Genomics (1 mentions)

Spelling variants of this product

Chromium Single Cell 3′ Library (33 citations) Chromium Single Cell 3′ v2 (4 citations) Chromium Single Cell 3′ v2 Library (2 citations)

9 protocols using chromium single cell 3 library kit v2

Single-cell RNA-seq of CD45+ Immune Cells

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Single-cell 3’ gene expression libraries for CD45⁺ immune cells were prepared by strictly following the protocols of a Chromium Single Cell 3’v2 Library kit (10x Genomics). All resulting libraries were sequenced on the Illumina NovaSeq6000 platform (Novogene and Berry Genomics, Beijing, China).

Zhou Z., Zhou X., Jiang X., Yang B., Lu X., Fei Y., Zhao L., Chen H., Zhang L., Si X., Liang N., Wang Y., Yang D., Peng Y., Yang Y., Yao Z., He Y., Wu X., Zhang W., Wang M., Yang H, & Zhang X. (2024). Single-cell profiling identifies IL1Bhi macrophages associated with inflammation in PD-1 inhibitor-induced inflammatory arthritis. Nature Communications, 15, 2107.

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Single-cell transcriptomics of mouse immune cells

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ILC2s were sorted from homogenized mouse lung, skin, and small intestine as live (DAPI⁻) CD45⁺Lin⁻(CD3,CD4,CD5,CD8α,CD11b,CD11c,CD19,NK1.1, NKp46,Gr-1,F4/80,Ter-119,DX5)Il5^RFP+ cells. BM cells were sorted as live CD45⁺Lin-Thy1⁺Arg1^YFP+ cells. Cells were sorted using a MoFlo XDP (Beckman Coulter) into ice-cold 0.5% BSA in PBS and processed on the same day through the Chromium Single Cell 3’ v2 Library Kit (10X Genomics) per the manufacturer’s protocol. Each channel was loaded with 5000–25000 cells from each tissue, yielding 400 single cells (neonatal small intestine), 3030 single cells (neonatal skin), 6348 single cells (neonatal lung), 7547 single cells (adult small intestine), 6115 single cells (adult skin), 11615 single cells (adult lung), and 4990 single cells (adult BM) for analysis. The cells were then partitioned into Gel Beads in Emulsion in the instrument, where cell lysis and barcoded reverse transcription of RNA occurred, followed by amplification, shearing, and 5’ adaptor and sample index sequence ligation. Libraries were sequenced on an Illumina HiSeq 4000. Single Cell 3’ libraries use standard Illumina sequencing primers for both sequencing and index reads. They were run using paired-end sequencing with single indexing where Read 1 is 26 cycles and Read 2 is 98 cycles.

Schneider C., Lee J., Koga S., Ricardo-Gonzalez R.R., Nussbaum J.C., Smith L.K., Villeda S.A., Liang H.E, & Locksley R.M. (2019). Tissue-resident group 2 innate lymphoid cells differentiate by layered ontogeny and in situ perinatal priming. Immunity, 50(6), 1425-1438.e5.

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Single-cell RNA-seq of IL31Ra-KO mouse skin

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HDM-treated dorsal neck skin from 4 pooled IL31RAKO and 4 pooled WT C57Bl/6 mice was digested and prepared for flow cytometry as above. The live (DAPI-negative) CD45 + cell fraction of each sample was sorted using a Moflo XDP (Beckman Coulter) directly into ice-cold 0.5% BSA in PBS, and immediately processed through the Chromium Single Cell 3′ v2 Library Kit (10X Genomics) per the manufacturer's protocol by the UCSF Genomics Core Facility of the UCSF Institute for Human Genetics. Each channel was loaded with 30,000 cells per sample. The cells were then partitioned into Gel Beads in Emulsion in the instrument, where cell lysis and barcoded reverse transcription of RNA occurred, followed by amplification, shearing, and 5′ adaptor and sample index attachment. Libraries were sequenced on an Illumina HiSeq 4000. Single Cell 3′ libraries used standard Illumina sequencing primers for both sequencing and index reads and were run using paired-end sequencing with single indexing where Read 1 is 26 cycles and Read 2 is 98 cycles.

Fassett M.S., Braz J.M., Castellanos C.A., Salvatierra J.J., Sadeghi M., Yu X., Schroeder A.W., Caston J., Munoz-Sandoval P., Roy S., Lazarevsky S., Mar D.J., Zhou C.J., Shin J.S., Basbaum A.I, & Ansel K.M. (2023). IL-31-dependent neurogenic inflammation restrains cutaneous type 2 immune response in allergic dermatitis. Science immunology, 8(88).

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Single-cell RNA-seq Library Preparation

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Single cells were purified by FACS sorting before library preparation and single-cell libraries were prepared with the Chromium Single Cell 3' Reagent Kits v2 (10x Genomics) as per manufacturer's instructions. Briefly, sorted cells in suspension were first prepared as gel beads in emulsion (GEMs) on Single Cell 3' Chips v2 (10x Chromium) using the Chromium Controller (10x Genomics). Barcoded RNA transcripts in each single cell were reverse transcribed within GEM droplets. cDNA was purified with DynaBeads MyOne Silane beads (Invitrogen) and then amplified for subsequent library construction. Sequencing libraries were prepared by fragmentation, end-repair, ligation with indexed adapters and PCR amplification using the Chromium Single Cell 3' library kit v2 (10x Genomics). Nucleic acid was cleaned up after each steps using SPRIselect beads (Beckman Coulter). Libraries were then quantified by Qubit and real-time quantitative PCR on a LightCycler 96 System (Roche).

Li J., Yang K.Y., Tam R.C., Chan V.W., Lan H.Y., Hori S., Zhou B, & Lui K.O. (2019). Regulatory T-cells regulate neonatal heart regeneration by potentiating cardiomyocyte proliferation in a paracrine manner. Theranostics, 9(15), 4324-4341.

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Single-Cell RNA-Seq of CD8+ T-Cells from Diabetic Mice

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CD45⁺CD3⁺CD8⁺ T-cells used for single-cell RNA-sequencing (scRNA-seq) were dissociated into single cells from the ischemic muscle of Lepr^db/+ or Lepr^db/db mice as aforementioned. Single-cell libraries were prepared with the Chromium Single Cell 3' Reagent Kits v2 (10x Genomics) as previously described 15 (link). Briefly, sorted single cell suspension was prepared as gel beads in emulsion (GEMs) on Single Cell 3' Chips v2 (10x Chromium) using the Chromium Controller (10x Genomics). Barcoded RNA transcripts in each single cell were reverse transcribed within GEM droplets. Complementary DNA (cDNA) was purified with DynaBeads MyOne Silane beads (Invitrogen) and then amplified for subsequent library construction. Sequencing libraries were prepared by fragmentation, end-repair, ligation with indexed adapters, and polymerase chain reaction (PCR) amplification using the Chromium Single Cell 3' library kit v2 (10x Genomics). Nucleic acid was cleaned up after each step using SPRIselect beads (Beckman Coulter). Libraries were then quantified by Qubit and real-time quantitative PCR on a LightCycler 96 System (Roche).

Liang C., Yang K.Y., Chan V.W., Li X., Fung T.H., Wu Y., Tian X.Y., Huang Y., Qin L., Lau J.Y, & Lui K.O. (2020). CD8+ T-cell plasticity regulates vascular regeneration in type-2 diabetes. Theranostics, 10(9), 4217-4232.

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Single-Cell RNA-Seq Library Preparation

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Single cells were purified by FACS sorting before library preparation, and single-cell libraries were prepared with the Chromium Single Cell 3’ Reagent Kits v2 (10x Genomics) as per manufacturer’s instructions. Briefly, sorted cells in suspension were first prepared as gel beads in emulsion (GEMs) on Single Cell 3’ Chips v2 (10x Chromium) using the Chromium Controller (10x Genomics). Barcoded RNA transcripts in each single cell were reverse transcribed within GEM droplets. cDNA was purified with DynaBeads MyOne Silane beads (Invitrogen) and then amplified for subsequent library construction. Sequencing libraries were prepared by fragmentation, end-repair, ligation with indexed adapters, and PCR amplification using the Chromium Single Cell 3’ library kit v2 (10x Genomics). Nucleic acid was cleaned up after each steps using SPRIselect beads (Beckman Coulter). Libraries were then quantified by Qubit and real-time quantitative PCR on a LightCycler 96 System (Roche).

Leung C.S., Yang K.Y., Li X., Chan V.W., Ku M., Waldmann H., Hori S., Tsang J.C., Lo Y.M, & Lui K.O. (2018). Single-cell transcriptomics reveal that PD-1 mediates immune tolerance by regulating proliferation of regulatory T cells. Genome Medicine, 10, 71.

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Single-cell RNA-seq Library Preparation

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Li J., Liang C., Yang K.Y., Huang X., Han M.Y., Li X., Chan V.W., Chan K.S., Liu D., Huang Z.P., Zhou B, & Lui K.O. (2020). Specific ablation of CD4+ T-cells promotes heart regeneration in juvenile mice. Theranostics, 10(18), 8018-8035.

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Single-cell RNA-seq Library Preparation

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To generate single-cell suspensions, cells were trypsinized as described. After stopping the reaction, cells were washed with PBS and filtered through a 40 µm cell strainer. Cells were washed, resuspended in PBS supplemented with 0.05% bovine serum albumin, and filtered through a 20 µm cell strainer. Single-cell suspensions were loaded following the Chromium Single Cell 3′ Library Kit v2 (10× Genomics, Pleasanton, CA, USA) protocol to generate cell and gel bead emulsions. Reverse transcription, cDNA amplification, and sequencing library generation were performed according to manufacturer’s protocol. Each library was sequenced in one lane of the NextSeq500 (Illumina, San Diego, CA, USA; high-output mode, paired-end, 26 × 49 bp).

Zowada M.K., Tirier S.M., Dieter S.M., Krieger T.G., Oberlack A., Chua R.L., Huerta M., Ten F.W., Laaber K., Park J., Jechow K., Müller T., Kalxdorf M., Kriegsmann M., Kriegsmann K., Herbst F., Krijgsveld J., Schneider M., Eils R., Glimm H., Conrad C, & Ball C.R. (2021). Functional States in Tumor-Initiating Cell Differentiation in Human Colorectal Cancer. Cancers, 13(5), 1097.

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Single-Cell RNA Sequencing of BM Cells

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Frozen BM cells were rapidly thawed, washed, counted and resuspended in PBS and 0.04% bovine serum albumin to a final concentration of 1000 cells per µl. The Chromium Controller (10x Genomics) was used for parallel sample partitioning and molecular barcoding. To generate a single-cell Gel Bead in Emulsion, cellular suspensions were loaded on a Single Cell 3′ chip together with the Single Cell 3′ Gel Beads, according to the manufacturer’s instructions (10x Genomics). scRNA-seq libraries were prepared using the Chromium Single Cell 3′ Library Kit v.2 (10x Genomics). Fourteen cycles were used for the total complementary DNA amplification reaction and for the total sample index PCR. Generated libraries were combined according to Illumina specifications and paired-end sequenced on HiSeq 2500/4000 platforms with standard Illumina sequencing primers for both sequencing and index reads; 100 cycles were used to sequence Read1 and Read2.

Boiarsky R., Haradhvala N.J., Alberge J.B., Sklavenitis-Pistofidis R., Mouhieddine T.H., Zavidij O., Shih M.C., Firer D., Miller M., El-Khoury H., Anand S.K., Aguet F., Sontag D., Ghobrial I.M, & Getz G. (2022). Single cell characterization of myeloma and its precursor conditions reveals transcriptional signatures of early tumorigenesis. Nature Communications, 13, 7040.

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