Chromium next gem single cell 5 library and gel bead kit v1

Manufactured by 10x Genomics

The Chromium Next GEM Single Cell 5' Library and Gel Bead Kit v1.1 is a laboratory equipment product designed for single-cell RNA sequencing. It provides the necessary reagents and components to generate single-cell gene expression libraries from individual cells.

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Lab products found in correlation

Chromium chip g, by 10x Genomics (2 mentions) Easysep dead cell removal kit, by STEMCELL (1 mentions) Hiseq x, by Illumina (1 mentions) Hiseq 2500, by Illumina (1 mentions) Facsaria 3, by BD (1 mentions) Anti cd28 clone cd28, by BD (1 mentions) Chromium single cell 5 d j enrichment kit for human t cells, by 10x Genomics (1 mentions) Facsmelody, by BD (1 mentions) Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions) Countess 2 fl, by Thermo Fisher Scientific (1 mentions) Totalseq c anti human hashtag antibodies, by BioLegend (1 mentions) Novaseq 6000, by Illumina (1 mentions) 7 aad, by BD (1 mentions) Anti mouse cd16 cd32 antibody, by Thermo Fisher Scientific (1 mentions) Anti cd45 bv510, by BioLegend (1 mentions) Gentlemacs octo dissociator, by Miltenyi Biotec (1 mentions) Tumor dissociation kit, by Miltenyi Biotec (1 mentions) Macsquant tyto, by Miltenyi Biotec (1 mentions) Novaseq 6000 machine, by Illumina (1 mentions)

5 protocols using chromium next gem single cell 5 library and gel bead kit v1

Mouse Lung Single-Cell RNA-Seq

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Lung single cell suspensions were prepared from 10 mice as described
above. Dead cells were removed with an EasySep Dead Cell Removal Kit
(Annexin V; StemCell). Cells were then stained with 1 μg TotalSeq-C
hashtag antibodies 1–10 (M1/42, 30-F11; Biolegend) and surface
antibodies against CD44 (IM7), CD8ɑ (53–6.7) and
H-2K^b-SIINFEKL and H-2K^b-SIYRYYGL tetramers for 30
minutes on ice. FACS sorting was performed on a FACS Aria III (BD
Biosciences) and CD44+ CD8α+ SIINFEKL+ and CD44+ CD8α+
SIYRYYGL+ T cells were sorted into separate tubes, counted and resuspended
in PBS (no calcium or magnesium) with 0.04% BSA. Approximately 65,000
SIINFEKL+ cells and 14,000 SIYRYYGL+ cells were loaded across two channels
of a Chromium single-cell 5’ chip (10X Genomics) according to
manufacturer’s instructions. Single cells were partitioned into
droplets with gel beads to form emulsions, after which cellular lysis and
barcoded reverse transcription of mRNA was performed. Paired 5’ gene
expression, hashtag barcode and V(D)J libraries were prepared using the
following kits from 10X Genomics and protocols provided by the manufacturer:
Chromium Next GEM Single Cell 5′ Library and Gel Bead Kit v1.1;
Chromium Single Cell V(D)J Enrichment Kit, Mouse T Cell. RNA expression
libraries were sequenced individually using HiSeq X (Illumina). VDJ and
hashtag barcode libraries were sequenced with HiSeq 2500 (Illumina).

Burger M.L., Cruz A.M., Crossland G.E., Gaglia G., Ritch C.C., Blatt S.E., Bhutkar A., Canner D., Kienka T., Tavana S.Z., Barandiaran A.L., Garmilla A., Schenkel J.M., Hillman M., de los Rios Kobara I., Li A., Jaeger A.M., Hwang W.L., Westcott P.M., Manos M.P., Holovatska M.M., Hodi F.S., Regev A., Santagata S, & Jacks T. (2021). Antigen Dominance Hierarchies Shape TCF1+ Progenitor CD8 T Cell Phenotypes in Tumors. Cell, 184(19), 4996-5014.e26.

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Single-cell analysis of SARS-CoV-2 vaccine-elicited T cells

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For single-cell RNA sequencing, PBMC of three BNT-BNT-BNT-vaccinated, three AZ-BNT-BNT vaccinated, and three BNT-BNT-infected donors were stimulated with 1 µg/ml S-I peptide pool in the presence of purified anti-CD28 (clone CD28.2, BD Biosciences) and anti-CD40 (clone HB14, Miltenyi Biotec). CD4⁺ T cells were enriched by MACS (Miltenyi Biotec) and CD40L⁺4-1BB⁺ CD4⁺ T cells FACS sorted using an FACS Melody (BD). The cells were loaded with a maximum concentration of 1000 cells/µl and a maximum cell number of 17.000 cells on a Chromium Chip G (10x Genomics). Gene expression and TCR libraries were generated according to the manufacturer’s instruction using the Chromium Next GEM single cell 5’Library and Gel bead Kit V1.1 and Chromium Single Cell V(D)J Enrichment Kit for human T cells (10x Genomics). Sequencing was conducted with a NovaSeq 6000 cartridge (Illumina) with 20.000 reads per cell for GEX libraries and 5.000 reads per cell for TCR libraries.

Henze L., Braun J., Meyer-Arndt L., Jürchott K., Schlotz M., Michel J., Grossegesse M., Mangold M., Dingeldey M., Kruse B., Holenya P., Mages N., Reimer U., Eckey M., Schnatbaum K., Wenschuh H., Timmermann B., Klein F., Nitsche A., Giesecke-Thiel C., Loyal L, & Thiel A. (2023). Primary ChAdOx1 vaccination does not reactivate pre-existing, cross-reactive immunity. Frontiers in Immunology, 14, 1056525.

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CITE-seq Analysis of PBMC from RV254 Participants

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Total PBMC from whole blood obtained from 18 RV254 participants 48 weeks after ART initiation were processed for CITE-seq on the 10x Genomics platform as described previously (Shangguan et al., 2021 ). Briefly, cell counts and viability were assessed by both the Countess II FL (Thermo Fisher) and trypan blue staining. Donor cells were hashed with TotalSeq-C anti-human Hashtag antibodies (BioLegend) and stained with a cocktail comprised of 63 TotalSeq-C antibodies targeting surface-expressed proteins. Cells from batches consisting of 9 or 10 samples were loaded into each of four Chromium chip wells before loading into the Chromium instrument. Cell mRNA gene expression (GEX) and antibody-derived tag (ADT) libraries were subsequently generated using the Chromium Next GEM Single Cell 5’ Library and Gel Bead Kit v1.1 (10x Genomics) according to the manufacturer’s instructions. Pooled libraries were quantitated with the MiSeq Nano v2 reagent cartridge (Illumina) and sequenced on an Illumina NovaSeq 6000 using an S4 reagent cartridge (2×100 bp)(Illumina). A modified version of the scRNA-seq assay was performed in RV254 participants at the acute HIV time point (n = 6) without feature barcodes.

Li S.S., Hickey A., Shangguan S., Ehrenberg P.K., Geretz A., Butler L., Kundu G., Apps R., Creegan M., Clifford R.J., Pinyakorn S., Eller L.A., Luechai P., Gilbert P.B., Holtz T.H., Chitwarakorn A., Sacdalan C., Kroon E., Phanuphak N., de Souza M., Ananworanich J., O’Connell R.J., Robb M.L., Michael N.L., Vasan S, & Thomas R. (2022). HLA-B*46 associates with rapid HIV disease progression in Asian cohorts and prominent differences in NK cell phenotype. Cell host & microbe, 30(8), 1173-1185.e8.

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Single-cell analysis of tumor immune landscape

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Four different tumors per regimen and per time point were dissociated to single-cell suspensions using a tumor dissociation kit (Miltenyi Biotech) and gentleMACS™ Octo Dissociator (Miltenyi Biotech). 5×10⁵ cells per tumor were pooled together (2×10⁶ in total) as one sample for each regimen and each time point. Cells were incubated with 20% FBS in PBS with 25 μg/mL of anti-mouse CD16/CD32 antibody (eBioscience) at 4°C for 10 min to minimize background antibody binding. Then cells were stained with BV510-anti-CD45 (1 μg/mL, Biolegend) and PerCP-anti-TER119 (2 μg/mL, Biolegend) at room temperature for 20 minutes, followed by 7AAD (10 μL in 500 μL PBS per sample, BD Pharmigen) staining for 5 minutes on ice. Cells after staining were sorted by BD FACSAria II sorting system to harvest the BV510 (CD45) positive and PerCP (TER119, 7AAD) negative population as live CD45⁺ cells. Cells recovered were subjected to 10X Genomics standard protocol for coupled scRNA-seq and scTCR-seq library preparation using Chromium Next GEM Single Cell 5′ Library and Gel Bead Kit v1.1 (10X Genomics) and V(D)J Enrichment Kit for Mouse T Cells (10X Genomics). Libraries were sequenced by Novaseq 6000 S2 flow cell with 2×50 reads targeting a minimum of 20,000 read pairs per cell for scRNA-seq library and 5,000 read pairs per cell for scTCR-seq library.

Wang Y., Liu S., Yang Z., Algazi A.P., Lomeli S.H., Wang Y., Othus M., Hong A., Wang X., Randolph C.E., Jones A.M., Bosenberg M.W., Byrum S.D., Tackett A.J., Lopez H., Yates C.C., Solit D.B., Ribas A., Piva M., Moriceau G, & Lo R.S. (2021). Anti-PD-1/L1 lead-In before MAPK inhibitor combination maximizes antitumor immunity and efficacy. Cancer cell, 39(10), 1375-1387.e6.

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Single-Cell Transcriptomics of CD154+ T Cells

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For single cell transcriptomics, CD154+ cells were isolated by MACS and further purified by FACS sorting on a MACSQuant Tyto (Miltenyi Biotec) based on dual expression of CD154 and CD69. Sorted CD154+ T cells were removed from the sorting chamber into pre-coated low-bind collection tubes, 1ml RPMI1640 medium supplemented with 5% AB Serum was added, and cells were centrifuged for 5 min at 400 x g, 4°C. The supernatant was carefully removed leaving 10-30 μL to reach a maximum concentration of 1000 cells /μl.
Single-cell suspensions were loaded on a Chromium Chip G (10x Genomics) according to the manufacturer’s instructions for processing with the Chromium Next GEM Single Cell 5¢ Library and Gel Bead Kit v1.1. Depending on the number of cells available for each patient, a maximum of 30,000 cells were loaded for each reaction. TCR single-cell libraries were subsequently prepared from the same cells with the Chromium Single Cell V(D)J Enrichment Kit, Human T Cell. Libraries were sequenced on Illumina NovaSeq 6000 machine with 2x100 bp for gene expression, aiming for 50,000 reads per cell and 2x150 bp and 5000 reads per cell for TCR libraries.

Bacher P., Rosati E., Esser D., Martini G.R., Saggau C., Schiminsky E., Dargvainiene J., Schröder I., Wieters I., Khodamoradi Y., Eberhardt F., Vehreschild M.J., Neb H., Sonntagbauer M., Conrad C., Tran F., Rosenstiel P., Markewitz R., Wandinger K.P., Augustin M., Rybniker J., Kochanek M., Leypoldt F., Cornely O.A., Koehler P., Franke A, & Scheffold A. (2020). Low-Avidity CD4+ T Cell Responses to SARS-CoV-2 in Unexposed Individuals and Humans with Severe COVID-19. Immunity, 53(6), 1258-1271.e5.

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