description: 3 pooled female mouse spleens
attributes.tag: strain
attributes.value: CFW (Swiss Webster)
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: breed
attributes.value: Outbred
attributes.tag: cultivar
attributes.value: not applicable
attributes.tag: ecotype
attributes.value: not applicable
attributes.tag: age
attributes.value: 10 weeks
attributes.tag: sex
attributes.value: female
attributes.tag: tissue
attributes.value: Spleen
attributes.tag: biomaterial_provider
attributes.value: Charles River Laboratories
attributes.tag: collection_date
attributes.value: 2020-09-28
attributes.tag: geo_loc_name
attributes.value: not applicable
attributes.tag: health_state
attributes.value: Healthy
attributes.tag: lat_lon
attributes.value: not applicable
attributes.tag: treatment
attributes.value: Solvent (7% Tween-80, 3% Ethanol, 90% diH2O) via P.O. admin route 24hr before organ harvest
attributes.tag: env_broad_scale
attributes.value: not applicable
attributes.tag: env_local_scale
attributes.value: not applicable
attributes.tag: env_medium
attributes.value: not applicable
attributes.tag: isol_growth_condt
attributes.value: not applicable
attributes.tag: ploidy
attributes.value: diploid
attributes.tag: samp_collect_device
attributes.value: Sterile surgical scissors and sterile tweezers
attributes.tag: samp_mat_process
attributes.value: Mechanical disruption to generate single-cell splenocyte suspension via rubber syringe plunger through 70-micron MACS strainer. RBC Lysis using Hybri-Max RBC lysis solution (45 secs). Depleted more prevalent cell types in the spleen in order to capture scRNA-seq of untouched (via negative immunomagnetic depletion) less prevalent cell types. This was executed using PE-conjugated antibodies to label cells for selective depletion (anti-CD3e-PE (T cells), anti-Ter119-PE (RBCs), anti-CD11c-PE (dendritic cells), anti-CD335-PE (NK cells), anti-Ly6G-PE (neutrophils), and anti-CD115-PE (monocytes)). Miltenyi immunomagnetic beads that were anti-PE and anti-CD19 were used to deplete the previously antigen-specific PE-labeled cells and B cells, respectively. Immunomagnetic depletion using LD columns with the QuadroMACS magnetic isolation system was used (Miltenyi Biotec) resulted in decreased major spleen cell populations which enriched rarer cell types (i.e., basopohils and eosinophils). A spiked sample was created by adding untouched splenocytes (33% of the final cell population) to the resulting enriched fraction (67% of the final cell population). This spiked sample was then analyzed by flow cytometry to confirm desired cell populations for broadened scRNA-seq. This spiked sample was subsequently used to produce a scRNA-seq data set using the 10X Genomics 3' scRNA-seq in accordance with the methods described in the SRA Metadata, "Design description" for library sequencing prep.
attributes.tag: samp_size
attributes.value: 3 pooled spleens
attributes.tag: BioSampleModel
attributes.value: Model organism or animal