Mixture of Cells from Mouse Lymph Nodes and Spleen Stained with TotalSeq™-C Mouse Universal Cocktail
Single Cell Gene Expression Flex dataset analyzed using Cell Ranger 7.2.0
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Spleen and abdominal lymph nodes were isolated from an 8 month old male C57BL/6 mouse.
A single cell suspension was prepared by mashing the tissues on a 70µm filter. Red blood cells were eliminated with eBioscience 1X RBC-lysis buffer (REF 00-4333-57) and passed through a 40µm filter. Cells were stained with TotalSeq™-C Mouse Universal Cocktail, V1.0 (BioLegend, Cat# 199903) following the demonstrated protocol Cell Surface Protein Labeling for Chromium Fixed RNA Profiling (CG000529, Rev B). True-Stain Monocyte Blocker (BioLegend, Cat# 426101) was added during incubation with TruStain FcX™ PLUS (anti-mouse CD16/32, BioLegend, Cat# 156604) After staining, cells were washed using the 1-Wash option before proceeeding with fluorescence activated cell sorting (FACS) to eliminate dead cells using the viability dye 7-AAD. Approximately 300,000 7-AAD-negative cells were then fixed for 1 hour at room temperature following (20°C) the demonstrated protocol Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling (CG000478).
The Fixed RNA Gene Expression library was generated as described in the Chromium Fixed RNA Profiling - Multiplexed Samples with Feature Barcode technology for Protein using Barcode Oligo Capture User Guide (CG000673, Rev A). The fixed cells were run as a multiplexed experiment, where two unique pairs of Probe Barcodes and Antibody Multiplexing Barcodes were used for each tissue type. After hybridization, cells were washed, and then run in a single GEM lane. The resulting libraries were sequenced on an Illumina NovaSeq6000 with approximately 32k read pairs per cell for Gene Expression library and 43k read pairs per cell for Protein Expression library.
- 28,945 cells detected (Lymph node rep 1: 7,579; Lymph node rep 2: 6,002; Spleen rep 1: 7,810; Spleen rep 2: 7,554)
- Paired-end, dual indexing: 28 cycles Read 1, 10 cycles i7, 10 cycles i5, 90 cycles Read 2
This dataset is licensed under the Creative Commons Attribution license.