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Status |
Public on Nov 09, 2022 |
Title |
Multiplexed CD40 liver experiment |
Sample type |
SRA |
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Source name |
Liver leucocytes
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Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 cell type: Liver leucocytes treatment: Anti-CD40 ((InVivoPlus BP0016-2) or saline (control mouse) culture supplement: -
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Treatment protocol |
Mice were injected with 20 mg/kg anti-CD40 antibody (InVivoPlus BP0016-2) intraveinously or saline (Control mouse).
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Growth protocol |
Specific-pathogen-free (SPF) housed 7-10 week old C57BL/6J (JAXTM strain) mice obtained from Charles River Laboratories were used for the experiment.
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Extracted molecule |
total RNA |
Extraction protocol |
At 7, 14 or 22 hours post injection, mice were euthanized and their liver digested into a non-parenchymal single-cell suspension. Cells were enriched for leucocytes using CD45+ coated magnetic beads (MagniSort™ Mouse CD45 Positive Selection Kit, 8802-6865-74), stained with Totalseq B hashtag antibodies (Biolegend) and pooled together at equal cell count before processing for single-cell RNA sequencing. Cells were processed according to the Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) with Feature Barcoding technology for Cell Surface Protein. For all experiments, the sample volume was adjusted to a target capture of 10,000 cells and loaded on the 10x Genomics chromium next-GEM chip G to generate gel-beads-in-emulsion (GEMs). The GEM solution was placed in a Applied Biosystems Veriti 96 well thermal cycler for reverse transcription as described by the 10x Genomics instruction guide (53:00 min at 53°C followed by 5:00 min at 85°C). The resulting barcoded cDNA was then cleaned using Dynabeads MyOne Silane and amplified for 11 cycles (as recommended by the 10x Genomics user guide for a target cell recovery of >6,000 cells). After amplification, for multiplexed experiments, cDNA generated from polyadenylated mRNA for the 3’ gene expression library was separated from DNA from the Cell Surface Protein Feature Barcode for the Cell Surface Protein library with Dynabeads MyOne Silane and SPRIselect reagents based on size. The quality and concentration of both cDNA and DNA were assessed using High-Sensitivity D5000 ScreenTape (Agilent). All samples presented product sizes with a narrow distribution centered around 2000 pb and yielded between 50 and 800 ng cDNA (manually selecting products between 100-250 and 5000-6000 bp). cDNA and DNA were then subjected to enzymatic fragmentation, end repair and A-tailing. Adaptors were ligated to the fragmented cDNA and DNA, and the sample index was added during sample index PCR (set for 12 cycles, as recommended by the 10x Genomics user guide to correlate with a cDNA/DNA input of 12-150 ng). Library quality and concentration were assessed using High-Sensitivity D5000 ScreenTape (Agilent). All gene expression libraries showed an average fragment size of around 400 pb. For multiplexed runs, 3ʹ Gene Expression and Cell Surface Protein libraries were pooled at a ratio of 4:1 and sequenced using the Illumina NovaSeq 6000 system with a sequencing depth of 50,000 and 12,500 reads per cell, respectively, following the recommendations of 10X Genomics (paired-end reads, single indexing, read 1 = 28 cycles, i7 = 8 cycles, i5 = 0 cycles and read 2 = 91 cycles). For non multiplexed runs, 3ʹ Gene Expression libraries for each sample were pooled at an equimolar amount and sequenced using the Illumina NovaSeq 6000 system with a sequencing depth of 50,000 reads per cell, following the same recommendations of 10x Genomics.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic single cell |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
multiplexed experiment with the following ADT_classification allocation to treatment: - Ctrl.1 (B0301-TotalSeq) = Control (saline) - CD40 7h (B0302-TotalSeq) = CD40 7h - CD40 14h (B0303-TotalSeq) = CD40 14h - CD40 22h (B0303-TotalSeq) = CD40 22h
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Data processing |
The Cell Ranger Single-Cell Software Suite (version 4.0.0) was used for cDNA oligopeptide alignment, barcode assignment and UMI counting from fastq files obtained from the Illumina sequencing. For each sample, the cell-containing droplets were filtered from the empty droplets, and this step was followed by the generation of an expression matrix using Cell Ranger Count (version 4.0.0). Demultiplexing of the cells within each sample was performed with the filtered matrix produced by Cell Ranger in R (version 4.4) using Seurat (version 3.2.3) (Hao et al. 2021) and the HTODemux function (positive quantile set at 0.99). The resulting gene expression matrices were further analyzed with Python (version 3.8.8) using the Scanpy (version 1.7.0) (Wolf et al. 2018) library. Low-quality cells were defined as expressing more than 5000 or less than 500 genes, more than 40% proportion of mitochondrial genes or more than 30% ribosomal genes, and excluded from downstream analysis. Genes expressed in less than 20 cells were also removed. Normalization was performed using the pool-based size factor estimation implemented in the scran R package (version 1.14.1)(Lun et al. 2016; Lun et al. 2016). Size factors were determined using the function and normalization was performed by dividing in each cell the total gene count by the cell specific size-factor, deconvoluted from the pool size factor. Data was log transformed. Principal component analysis (PCA) was performed using the tl.pca function (Scanpy 1.7.0) with default settings followed by nearest neighbor graph construction using the pp.neighbors (Scanpy 1.7.0) function with the first 15 PCAs. Further dimension reduction was performed using the Leiden algorithm and cells plotted in 2 dimensions using UMAP plots. Differential gene expression was calculated using the tl.rank_genes_group function (Scanpy 1.7.0) or DEseq2 (1.26.0) (Love et al. 2014) implemented in the FindMarkers function of the Seurat package (3.2.3). Assembly: Ensembl GRCm38.p6 Release M23
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Submission date |
May 12, 2022 |
Last update date |
Nov 09, 2022 |
Contact name |
Marc Pfefferle |
E-mail(s) |
marc.pfefferle@uzh.ch
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Organization name |
University hospital Zürich (USZ)
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Street address |
Wagistrasse 12
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City |
Schlieren |
State/province |
Schweiz |
ZIP/Postal code |
8952 |
Country |
Switzerland |
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Platform ID |
GPL24247 |
Series (2) |
GSE202916 |
Targeted erythrophagocyte reprogramming of Kupffer cells halts cancer immunotherapy associated liver toxicity [Multiplexed_CD40] |
GSE202918 |
Targeted erythrophagocyte reprogramming of Kupffer cells halts cancer immunotherapy associated liver toxicity |
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Relations |
BioSample |
SAMN28215403 |
SRA |
SRX15246006 |
Supplementary file |
Size |
Download |
File type/resource |
GSM6138431_multiplexed_CD40_timepoint.h5ad.gz |
48.3 Mb |
(ftp)(http) |
H5AD |
GSM6138431_multiplexed_CD40_timepoint_ADT_classification.tsv.gz |
92.4 Kb |
(ftp)(http) |
TSV |
GSM6138431_multiplexed_CD40_timepoint_ADT_classification_global.tsv.gz |
83.3 Kb |
(ftp)(http) |
TSV |
GSM6138431_multiplexed_CD40_timepoint_CellCyclePhase.txt.gz |
198.4 Kb |
(ftp)(http) |
TXT |
GSM6138431_multiplexed_CD40_timepoint_barcodes.tsv.gz |
80.4 Kb |
(ftp)(http) |
TSV |
GSM6138431_multiplexed_CD40_timepoint_features.tsv.gz |
225.3 Kb |
(ftp)(http) |
TSV |
GSM6138431_multiplexed_CD40_timepoint_matrix.mtx.gz |
54.7 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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