Single cell 5 feature barcode library kit

Manufactured by 10x Genomics

The Single Cell 5' Feature Barcode Library Kit is a laboratory equipment product designed for the preparation of sequencing libraries for single-cell RNA sequencing analysis. The kit provides the necessary reagents and protocols to generate libraries containing cellular barcodes and unique molecular identifiers, which are essential for the identification and quantification of individual transcripts in single-cell samples.

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Lab products found in correlation

Novaseq 6000, by Illumina (3 mentions) Totalseq c antibody cocktail, by BioLegend (2 mentions) Fragment analyzer, by Agilent Technologies (1 mentions) Nextseq 500 device, by Illumina (1 mentions) Chromium single cell 5 library gel bead kit, by 10x Genomics (1 mentions) Qubit dna hs assay kit, by Thermo Fisher Scientific (1 mentions) Hs ngs fragment kit 1 6000bp, by Agilent Technologies (1 mentions)

Spelling variants of this product

5′ Feature Barcode Kit (6 citations) Chromium Single Cell 5′ Feature Barcode Library Kit (4 citations)

5 protocols using single cell 5 feature barcode library kit

Single-Cell Genomics and Immune Profiling

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Single cell suspensions were obtained by cell sorting and applied to the 10x Genomics workflow for cell capturing and scRNA gene expression (GEX) and TCR/BCR/CiteSeq library preparation using the Chromium Single Cell 5′ Library & Gel Bead Kit as well as the Single Cell 5′ Feature Barcode Library Kit (10x Genomics). After cDNA amplification the CiteSeq libraries were prepared separately using the Single Index Kit N Set A. TCR/BCR target enrichment was performed using the Chromium Single Cell V(D)J Enrichment Kit for Human T cells and B cells, respectively. Final GEX and TCR/BCR libraries were obtained after fragmentation, adapter ligation, and final Index PCR using the Single Index Kit T Set A. Qubit HS DNA assay kit (Life Technologies) was used for library quantification and fragment sizes were determined using the Fragment Analyzer with the HS NGS Fragment Kit (1–6000 bp) (Agilent).
Sequencing was performed on a NextSeq500 device (Illumina) using High Output v2 Kits (150 cycles) with the recommended sequencing conditions for 5′ GEX libraries (read1: 26nt, read2: 98nt, index1: 8nt, index2: n.a.) and Mid Output v2 Kits (300 cycles) for TCR/BCR libraries (read1: 150nt, read2: 150nt, index1: 8nt, index2: n.a., 20% PhiX spike-in).

Ferreira-Gomes M., Kruglov A., Durek P., Heinrich F., Tizian C., Heinz G.A., Pascual-Reguant A., Du W., Mothes R., Fan C., Frischbutter S., Habenicht K., Budzinski L., Ninnemann J., Jani P.K., Guerra G.M., Lehmann K., Matz M., Ostendorf L., Heiberger L., Chang H.D., Bauherr S., Maurer M., Schönrich G., Raftery M., Kallinich T., Mall M.A., Angermair S., Treskatsch S., Dörner T., Corman V.M., Diefenbach A., Volk H.D., Elezkurtaj S., Winkler T.H., Dong J., Hauser A.E., Radbruch H., Witkowski M., Melchers F., Radbruch A, & Mashreghi M.F. (2021). SARS-CoV-2 in severe COVID-19 induces a TGF-β-dominated chronic immune response that does not target itself. Nature Communications, 12, 1961.

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CITE-seq Analysis of COVID-19 and Healthy PBMC Samples

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CITE-seq data were generated from frozen PBMCs of 36 COVID-19 patients and 11 healthy controls as described by Stephenson et al.⁷ Briefly, after thawing, pools of 4 samples were generated by combined 500,000 viable cells per individual (total of 2 million cells per pool). TotalSeq-C™ antibody cocktail (BioLegend 99813) was used to perform cell surface marker staining on 500,000 cells per pool. 50,000 live cells (up to a maximum of 60,000 total cells) for each pool were processed using Single Cell V(D)J 5’ version 1.1 (1000020) together with Single Cell 5’ Feature Barcode library kit (1000080), Single Cell V(D)J Enrichment Kit, Human B Cells (1000016) and Single Cell V(D)J Enrichment Kit, Human T Cells (1000005) (10xGenomics) according to the manufacturer's protocols. Samples were sequenced on NovaSeq 6000 (Illumina) using S1 flowcells. Droplet libraries were processed using Cellranger v4.0. Reads were aligned to the GRCh38 human genome concatenated to the SARS-Cov-2 genome (NCBI SARS-CoV-2 isolate Wuhan-Hu-1) using STAR¹⁸ (link) and unique molecular identifiers (UMIs) deduplicated. CITE-seq UMIs were counted for GEX and ADT libraries simultaneously to generate feature X droplet UMI count matrices.

Kotagiri P., Mescia F., Hanson A.L., Turner L., Bergamaschi L., Peñalver A., Richoz N., Moore S.D., Ortmann B.M., Dunmore B.J., Morgan M.D., Tuong Z.K., Göttgens B., Toshner M., Hess C., Maxwell P.H., Clatworthy M.R., Nathan J.A., Bradley J.R., Lyons P.A., Burrows N, & Smith K.G. (2022). The impact of hypoxia on B cells in COVID-19. EBioMedicine, 77, 103878.

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Single-Cell Analysis of Human B and T Cells

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50,000 live cells (up to a maximum of 60,000 total cells) for each pool were processed using Single Cell VDJ 5′ version 1.1 (1000020) together with Single Cell 5′ Feature Barcode library kit (1000080), Single Cell V(D)J Enrichment Kit, Human B Cells (1000016) and Single Cell V(D)J Enrichment Kit, Human T Cells (1000005) from 10X Genomics following manufacturer’s recommendations. The samples were subjected to 12 cycles of cDNA amplification and 8 cycles for the protein library construction. The rest of libraries were processed as indicated by the manufacturer.
Libraries were pooled per sample using the ratio 9:2.4:1:0.6 for gene expression, feature barcoding, TCR enriched and BCR enriched libraries.
Samples were sequenced in Illumina NovaSeq6000 sequencer machine using S1 flowcells.

Wang J., Kotagiri P., Lyons P.A., Al-Lamki R.S., Mescia F., Bergamaschi L., Turner L., Morgan M.D., Calero-Nieto F.J., Bach K., Mende N., Wilson N.K., Watts E.R., Maxwell P.H., Chinnery P.F., Kingston N., Papadia S., Stirrups K.E., Walker N., Gupta R.K., Menon D.K., Allinson K., Aitken S.J., Toshner M., Weekes M.P., Nathan J.A., Walmsley S.R., Ouwehand W.H., Kasanicki M., Göttgens B., Marioni J.C., Smith K.G., Pober J.S, & Bradley J.R. (2022). Coagulation factor V is a T-cell inhibitor expressed by leukocytes in COVID-19. iScience, 25(3), 103971.

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Single-cell analysis of antigen-specific B cells

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Two subjects were enrolled for serial weekly blood draws one week after IIV. B cells were negative selected using column purification (EasySep Pan-B cell purification kit, StemCell Technologies), treated with neuraminidase, stained with HA tetramers, fluorophore-labeled Abs, and anti-FcRL5 Ab conjugated with TotalSeq C oligomer hashtags (Table S4). HA-specific IgD^neg CD38^lo/med cells were sort-purified as described in Table S5 and bar coded. Single cell suspensions were applied to the 10XGenomics workflow for cell capture, scRNA gene expression (GEX), BCR and Feature Barcoding library preparation using the Chromium Single Cell 5’ Library and Gel Bead Kit (Nest GEM 5’ Kit v 1.1) as well as the Single Cell 5’ Feature Barcode Library Kit (10XGenomics), following manufacturer’s instructions.

Nellore A., Zumaquero E., Scharer C.D., Fucile C.F., Tipton C.M., King R.G., Mi T., Mousseau B., Bradley J.E., Zhou F., Mutneja S., Goepfert P.A., Boss J.M., Randall T.D., Sanz I., Rosenberg A.F, & Lund F.E. (2023). A transcriptionally distinct subset of Influenza-specific effector memory B cells predicts long-lived antibody responses to vaccination in humans. Immunity, 56(4), 847-863.e8.

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CITE-seq Protocol for Immune Profiling

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CITE-seq data were generated from frozen PBMCs as described by Stephenson et al. (Stephenson et al., 2021 (link)). Briefly, after thawing, pools of 4 samples were generated by combined 500,000 viable cells per individual (total of 2 million cells per pool). TotalSeq-C antibody cocktail (BioLegend 99813) was used to perform cell surface marker staining on 500,000 cells per pool. 50,000 live cells (up to a maximum of 60,000 total cells) for each pool were processed using Single Cell V(D)J 5¢ version 1.1 (1000020) together with Single Cell 5¢ Feature Barcode library kit (1000080), Single Cell V(D)J Enrichment Kit, Human B Cells (1000016) and Single Cell V(D)J Enrichment Kit, Human T Cells (1000005) (10xGenomics) according to the manufacturer’s protocols. Samples were sequenced on NovaSeq 6000 (Illumina) using S1 flowcells. Droplet libraries were processed using Cellranger v4.0. Reads were aligned to the GRCh38 human genome concatenated to the SARS-Cov-2 genome (NCBI SARS-CoV-2 isolate Wuhan-Hu-1) using STAR (Dobin et al., 2013 (link)) and unique molecular identifiers (UMIs) deduplicated. CITE-seq UMIs were counted for GEX and ADT libraries simultaneously to generate feature X droplet UMI count matrices.

Bergamaschi L., Mescia F., Turner L., Hanson A.L., Kotagiri P., Dunmore B.J., Ruffieux H., De Sa A., Huhn O., Morgan M.D., Gerber P.P., Wills M.R., Baker S., Calero-Nieto F.J., Doffinger R., Dougan G., Elmer A., Goodfellow I.G., Gupta R.K., Hosmillo M., Hunter K., Kingston N., Lehner P.J., Matheson N.J., Nicholson J.K., Petrunkina A.M., Richardson S., Saunders C., Thaventhiran J.E., Toonen E.J., Weekes M.P., Göttgens B., Toshner M., Hess C., Bradley J.R., Lyons P.A, & Smith K.G. (2021). Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease. Immunity, 54(6), 1257-1275.e8.

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