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Sample GSM6613425

Query DataSets for GSM6613425

Status

Public on Oct 05, 2022

Title

PBMCs, TSLP_CCL3, GEX

Sample type

SRA

Source name

PBMCs, TSLP_CCL3, 12 hours

Organism

Mus musculus

Characteristics

tissue: blood
strain: C57BL/6J
cell type: Peripheral blood mononuclear cells
genotype: wild-type
treatment: TSLP, CCL3

Treatment protocol

Mice were intravenously injected with 2.5 µg of 48 recombinant cytokines. Cytokine-injected mice were anesthetized with avertin (250-500 mg/kg) 12 hours after cytokine injection and whole blood was collected from the left ventricle of the heart into the tubes containing ice-cold buffer (PBS plus 0.5% FCS and 2 mM EDTA). Red blood cells were lysed in ACK Red blood cell lysis buffer (Lonza, BW10548E) for 3 min and then three biological replicates were pooled into a tube. After pooling samples, the cells were washed by the buffer. We next used anti-Ter119 MicroBeads (Miltenyi Biotec, 130-049-901) to deplete Ter119+ cells thoroughly. Cells were then labelled with the TotalSeqTM-C0301 anti-mouse Hashtag 1 antibody (BioLegend, 155861) and TotalSeqTM-C0302 anti-mouse Hashtag 2 antibody (BioLegend, 155863) at 0.25 µg/sample for 15 min at 4°C. After staining, cells were washed with PBS, and resuspended in PBS + 0.04% BSA. Cells were counted and resuspended at a density of 1000 cells/µl.

Extracted molecule

total RNA

Extraction protocol

Samples were immediately run on the 10X Chromium (10X Genomics)
Samples were processed through library preparation following the recommended protocol (CG000186 Rev A) for the Chromium Single Cell V(D)J Reagent Kit.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

NextSeq 550

Description

whole_cyto_normalized.h5ad
whole_cyto_raw.h5ad

Data processing

Raw sequencing data were processed by the Cell Ranger pipeline to produce the cell by gene matrix of UMI counts.
Alignments were demultiplexed with a custom script and hashtag indexes.
Quality control was performed to filter out cells with too low (< 400) or high (> 22,000) counts and cells with a large fraction of mitchondrial counts (> 20%), and filter out genes expressed in fewer than 20 cells.
Cells were clustered into different cell types based on transcriptional profiles using the Louvain algorithm.
Based on known marker genes, we annotated 8 major cell types: B cells (Cd19, Cd79a, Cd79b), CD4+ T cells (Cd3d, Cd3e, Thy1, Cd4), CD8+ T cells (Cd3d, Cd3e, Thy1, Cd8a), dendritic cells (Flt3), Ly6C- monocytes (Nr4a1, Pparg), Ly6C+ monocytes (Ly6c2, F13a1), neutrophils (S100a8, S100a9), and NK cells (Ncr1).
Assembly: mm10
Supplementary files format and content: h5ad files; data matrix of shape, annotation of variables/features, and annotation of observations
Supplementary files format and content: whole_cyto_raw.h5ad (the filtered counts data for GEX and Hashtag samples)
Supplementary files format and content: whole_cyto_normalized.h5ad (normalized, log-transformed data for GEX and Hashtag samples)
Supplementary files format and content: whole_cyto_annot.csv (Annotations for each of the cells (rows) in the table. Notable columns are the sample/cytokine they came from, and the assigned celltype.)

Submission date

Oct 02, 2022

Last update date

Oct 05, 2022

Contact name

Nicolas Chevrier

E-mail(s)

nchevrier@uchicago.edu

Organization name

The University of Chicago

Department

Pritzker School of Molecular Engineering