Chromium single cell human bcr amplification kit

Manufactured by 10x Genomics

The Chromium Single Cell Human BCR Amplification Kit is a laboratory equipment product designed for the amplification and analysis of B-cell receptor (BCR) sequences from individual cells. It enables the simultaneous capture and profiling of paired heavy and light chain variable region sequences from single cells.

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Lab products found in correlation

5 feature barcode kit, by 10x Genomics (4 mentions) Chromium next gem single cell 5 kit v2, by 10x Genomics (3 mentions) Chromium next gem chip k single cell kit, by 10x Genomics (2 mentions) Dual index kit tt set a, by 10x Genomics (2 mentions) Library construction kit, by 10x Genomics (2 mentions) Dual index kit tn set a, by 10x Genomics (2 mentions) Novaseq 6000, by Illumina (2 mentions) Qubit fluorometer, by Thermo Fisher Scientific (1 mentions) Novaseq 6000, by 10x Genomics (1 mentions) Facsaria 2, by BD (1 mentions) Facsdiva 8, by BD (1 mentions) Labchip gx touch, by PerkinElmer (1 mentions) Fragment analyzer, by Thermo Fisher Scientific (1 mentions) Chromium system, by 10x Genomics (1 mentions) Qubit 3, by Thermo Fisher Scientific (1 mentions) Novaseq 6000 sequencer, by Illumina (1 mentions) Sh800, by Sony (1 mentions) Chromium next gem single cell 5 reagent kit v2, by 10x Genomics (1 mentions) Nextseq 550, by Illumina (1 mentions)

7 protocols using chromium single cell human bcr amplification kit

Single-Cell Immune Profiling with Multiplexed Antibodies

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After being processed, the 3 immune cell population fractions were stained with TotalSeq-C Human Universal Cocktail, V1.0 (BioLegend, catalog 399905), as well as Total-Seq anti–human CD72, IgG Fc, CD197 (CCR7), CD45RB, CD193 (CCR3), TCR, LAIR1 (CD395), and CD366 (Tim-3) (BioLegend, catalogs 316211, 410727, 353251, 310211, 310733, 331231, 342807, and 345049, respectively). Cells were pooled and washed using the Laminar Wash Mini system (Curiox Biosystems) before using the Chromium Next GEM Single Cell 5’ Kit v2 and Chromium Next GEM Chip K Single Cell Kit (10X Genomics).
Libraries were created using the Library Construction Kit, 5’Feature Barcode Kit, Chromium Single Cell Human BCR Amplification Kit, Dual Index Kit TT Set A (PN-1000215), and Dual Index Kit TN Set A (PN-1000250) (10X Genomics).Their quality and quantity was assessed using the Agilent Tapestation system and the Qubit Fluorometer (Invitrogen), before they were sent for sequencing on Illumina NovaSeq 6000 as per the instructions provided in 10X Genomics user guide. Sequencing was performed by SNP&SEQ Technology Platform, Science for Life Laboratory (Uppsala Biomedical Centre, Uppsala University, Sweden).

Scharf L., Axelsson H., Emmanouilidi A., Mathew N.R., Sheward D.J., Leach S., Isakson P., Smirnov I.V., Marklund E., Miron N., Andersson L.M., Gisslén M., Murrell B., Lundgren A., Bemark M, & Angeletti D. (2023). Longitudinal single-cell analysis of SARS-CoV-2–reactive B cells uncovers persistence of early-formed, antigen-specific clones. JCI Insight, 8(1), e165299.

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Single-cell transcriptome and BCR profiling

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Synovial tissue samples were thawed and resuspended according to the 10X protocol as described above. Cells were washed and then stained in FACS buffer at 4°C for 20 minutes with fluorescently conjugated antibodies as described in Supplementary Table 3. Single CD19^+ve DAPI^-ve CD3^-ve CD14^-ve B cells were then sorted with the FACS Aria II and FACS Diva 8.0.1 (BD Biosciences). The full gating strategy is shown in Supplementary Figure S2A. Immediately after sorting the CD19^+ve B cells were processed through the Chromium Single Cell Platform using the Chromium Next GEM Single Cell 5’ Kit v2 (10X Genomics, PN- 1000265), the Chromium Single Cell Human BCR Amplification Kit (10X Genomics, PN-1000253), and the Chromium Next GEM Chip K Single Cell Kit (10X Genomics, PN-1000287) as per manufacturer’s protocol. GEX and V(D)J libraries were then sequenced using the NextSeq2000 at the Wellcome Trust Clinical Research Facility’s Genetics Core in Edinburgh.

Wing E., Sutherland C., Miles K., Gray D., Goodyear C.S., Otto T.D., Breusch S., Cowan G, & Gray M. (2023). Double-negative-2 B cells are the major synovial plasma cell precursor in rheumatoid arthritis. Frontiers in Immunology, 14, 1241474.

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Single-Cell Sequencing of Antigen-Specific B Cells

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10x Genomics single-cell immune profiling technology was used for single-cell sequencing of antigen-specific B cells. Single-cell suspensions were loaded onto the GEM channels of the Chromium Controller microfluidics device (10x Genomics) and processed following the manufacturer’s protocol for target cell recovery of at most 10,000 B cells. Single-cell 5′ RNA sequencing (RNA-seq) and BCR sequencing (BCR-seq) libraries were prepared using the Chromium Next GEM Single Cell 5′ Kit v2 (10x Genomics) and the Chromium Single Cell Human BCR Amplification Kit (10x Genomics), respectively. The antigen, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), and hashtag antibody libraries were prepared and amplified using a 5′ Feature Barcode Kit (10x Genomics). All library preparations were performed according to the manufacturer’s protocols, and the final libraries were assessed using a LabChip GX Touch (PerkinElmer) instrument to verify the cDNA fragment size. The 5′ RNA-seq, BCR-seq, and feature barcode libraries were analyzed, quantified, and sequenced using DNBSEQ-G400 (MGI). This resulted in approximately 50,000 reads per cell for each 5′ RNA-seq library.

Onodera T., Sax N., Sato T., Adachi Y., Kotaki R., Inoue T., Shinnakasu R., Nakagawa T., Fukushi S., Terooatea T., Yoshikawa M., Tonouchi K., Nagakura T., Moriyama S., Matsumura T., Isogawa M., Terahara K., Takano T., Sun L., Nishiyama A., Omoto S., Shinkai M., Kurosaki T., Yamashita K, & Takahashi Y. (2023). CD62L expression marks SARS-CoV-2 memory B cell subset with preference for neutralizing epitopes. Science Advances, 9(24), eadf0661.

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Multimodal Single-Cell Library Preparation

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VDJ, 5′-mRNA, and feature barcode libraries were prepared using the 10× Chromium System (10X Genomics, Pleasanton, CA) according to the manufacturer’s instructions. The C Chromium Next GEM Single Cell 5′ Kit v2 (10X Genomics, PN-1000263), Chromium Single Cell Human BCR Amplification Kit (10X Genomics, PN-1000253), and 5′ Feature Barcode Kit (10X Genomics, PN-1000256), Library Construction Kit (10X Genomics, PN-1000190), and Dual Index Kit TT Set A (10X Genomics, PN-1000215), Dual Index Kit TN Set A (10X Genomics, PN-1000250) were used. All libraries were quantified by using Qubit 3.0 (Thermo Fisher), Fragment Analyzer, and qPCR. Sequencing was performed on a Hiseq 2500 platform running Rapid SBS Kit V2 2x100bp kit (Illumina, FC-402–4021), with 10 cycles for the i7 index and i5 index. Average sequencing depth aimed for the mRNA library is 20,000 read pairs per cell, 5000 read pairs per cell for the VDJ libraries, and 5000 read pairs for feature barcode libraries.

Cao Y., Yisimayi A., Bai Y., Huang W., Li X., Zhang Z., Yuan T., An R., Wang J., Xiao T., Du S., Ma W., Song L., Li Y., Li X., Song W., Wu J., Liu S., Li X., Zhang Y., Su B., Guo X., Wei Y., Gao C., Zhang N., Zhang Y., Dou Y., Xu X., Shi R., Lu B., Jin R., Ma Y., Qin C., Wang Y., Feng Y., Xiao J, & Xie X.S. (2021). Humoral immune response to circulating SARS-CoV-2 variants elicited by inactivated and RBD-subunit vaccines. Cell Research, 31(7), 732-741.

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Single-cell analysis of COVID-19 PBMC

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PBMC samples recovered from mild, severe and critical COVID-19 patients were stained with anti-human CD45 antibody. Live CD45⁺ cells and CD45⁺CD19⁺ cells (3,000-10,000 cells each, cell viability >98%) were sorted using a cell sorter (SH800S; SONY) and then encapsulated into droplets for gene expression and BCR repertoire analyses. Libraries for the analyses were prepared using Chromium Single Cell 5’ Reagent Kits v1.1 and Chromium Single Cell Human BCR Amplification Kit by following the manufacturer’s protocol (10X Genomics). The generated scRNA-seq and sc V(D)J -seq libraries were sequenced using a total of 308 cycles (paired-end reads) with a NovaSeq 6000 sequencer (Illumina, Inc.).

Aoki A., Iwamura C., Kiuchi M., Tsuji K., Sasaki A., Hishiya T., Hirasawa R., Kokubo K., Kuriyama S., Onodera A., Shimada T., Nagaoka T., Ishikawa S., Kojima A., Mito H., Hase R., Kasahara Y., Kuriyama N., Nakamura S., Urushibara T., Kaneda S., Sakao S., Nishida O., Takahashi K., Kimura M.Y., Motohashi S., Igari H., Ikehara Y., Nakajima H., Suzuki T., Hanaoka H., Nakada T.A., Kikuchi T., Nakayama T., Yokote K, & Hirahara K. (2024). Suppression of Type I Interferon Signaling in Myeloid Cells by Autoantibodies in Severe COVID-19 Patients. Journal of Clinical Immunology, 44(4), 104.

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Single-Cell Sequencing of B Cells

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Single cell suspensions of each tissue were prepared as described above. B cells were enriched by fluorescence-associated cell sorting (FACS) using antibodies against CD45, CD33, CD66b, CD3 and CD19. Enrichment resulted in ~90% purity of live, CD45⁺ CD66b⁻, CD33⁻, CD3⁻, CD19⁺ cells from each tissue. Enriched B cell populations from each sample were resuspended in PBS FBS EDTA and stained with 3ul of a unique TotalSeq-C DNA-barcoded hashtag (Supplementary Table 4) for 30 minutes on ice. Cells were washed, counted and combined in PBS FBS ensuring equal numbers of live cells were present from each sample.
CITE/scBCR-seq libraries were constructed using the Chromium Next GEM Single Cell 5’ Reagent Kit v2 (10x Genomics) with both a 5’ Feature Barcode Kit (10x Genomics) for antibody-derived tag (ADT) amplification and a Chromium Single Cell Human BCR Amplification Kit (10x Genomics) for targeted BCR amplification, according to the manufacturer’s instructions. The ADT library pool and scRNA-seq library pool were each sequenced on one lane of an S4 flow cell on an Illumina NovaSeq 6000 (~2.5 billion 2×100 bp reads per lane). The scBCR-seq library pool was sequenced with a 150 cycle High-Output Kit on an Illumina NextSeq 550 (read 1: 26 bp, read 2: 114 bp, index read 1: 10 bp, index read 2: 10 bp).

Matsumoto R., Gray J., Rybkina K., Oppenheimer H., Levy L., Friedman L.M., Khamaisi M., Meng W., Rosenfeld A.M., Guyer R.S., Bradley M.C., Chen D., Atkinson M.A., Brusko T.M., Brusko M., Connors T.J., Prak E.T., Hershberg U., Sims P.A., Hertz T, & Farber D.L. (2023). Induction of bronchus-associated lymphoid tissue is an early life adaptation for promoting human B cell immunity. Nature immunology, 24(8), 1370-1381.

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Single Cell BCR Sequencing Protocol

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The concentration of the single cell suspension was counted and adjusted to 1000 cells/μl. The single cell suspensions were loaded onto the Chromium Controller microfluidics device (10X Genomics) and processed using Chromium Next GEM Single Cell 5’ Kits v2 according to manufacturer’s protocol. The remaining procedures, including library construction, were performed according to the protocols of the Chromium Single Cell Human BCR Amplification Kit (10X Genomics). Following library construction, the BCR libraries were sequenced on an Illumina platform (NovaSeq 6000) using 2×150bp kit.

Yang X., Zhu Y., Chen S., Zeng H., Guan J., Wang Q., Lan C., Sun D., Yu X, & Zhang Z. (2021). Novel Allele Detection Tool Benchmark and Application With Antibody Repertoire Sequencing Dataset. Frontiers in Immunology, 12, 739179.

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