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| Status |
Public on Jan 25, 2022 |
| Title |
single cell suspension positively enriched for macrophages using F4/80 magnetic beads from a spleen of a SCD mouse |
| Sample type |
SRA |
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| Source name |
spleen
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| Organism |
Mus musculus |
| Characteristics |
strain: homozygous for Hbatm1Paz, homozygous for Hbbtm1Tow, homozygous for Tg(HBA-HBBs)41Paz (-/-, -/-, Tg/Tg, sickling)= SCD mice
tissue: spleen
enrichment: single-cell suspensions from spleen positively enriched for macrophages using anti-F4/80 antibody (rat, Bioscience)-coated Dynabeads (anti-rat IgG, Invitrogen)
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| Treatment protocol |
no treatment
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| Growth protocol |
mice aged 12-16 weeks
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| Extracted molecule |
total RNA |
| Extraction protocol |
Spleens were harvested and mechanically disrupted in PBS and passed through a 70-μm cell strainer. The cell suspensions were then centrifuged (300 xg, 5 min, 4°C), incubated in RBC lysis buffer (BioLegend) for 2 min at 37°C and centrifuged once more to obtain spleen cell populations devoid of mature erythrocytes. Macrophages were positively enriched from single-cell suspensions of spleen using anti-F4/80 antibody (rat, Bioscience)-coated Dynabeads (anti-rat IgG, Invitrogen) . The enriched Macrophages populations from the spleen of SCD mice and control littermates were then processed according to the 10x Genomics Chromium Single Cell 3’ v3.1 Reagent Kits. 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 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. 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 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 |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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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 data 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) 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 1.6.0) library. Cells with a total number of expressed genes > 5,000 or < 500, a proportion of mitochondrial genes < 15%, or a proportion of ribosomal genes < 30% and genes expressed in < 20 cells were excluded from downstream analyses. After filtering, normalization was performed using the scran normalization algorithm implemented in the scran R package (version 1.18.3), which was followed by log normalization. For dimensional reduction, principal component analysis (PCA) of each sample was performed using the tl.pca function with the default settings from the Scanpy library. For cell culture and in vivo samples, a shared nearest neighbor graph was built using the pp.neighbors function based on the first 15 PCAs or first 30 PCAs, respectively. Using the Leiden algorithm, cells were clustered and visualized in 2D using UMAP. For expression intensity projections, the cells were colored according to their log-normalized gene expression. Differentially expressed genes (p-value < 0.05 and a log2-fold change > 0.5) were determined using the tl.rank_genes_groups function from the Scanpy library with a pairwise Wilcoxon rank-sum test.
Genome_build: Ensembl GRCm38.p6 Release M23
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| Submission date |
Jul 02, 2021 |
| Last update date |
Jan 25, 2022 |
| Contact name |
Raphael Matthias Buzzi |
| E-mail(s) |
raphael.buzzi@usz.ch
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| Organization name |
Universitätsspital and University of Zurich
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| Department |
Division of Internal Medicine
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| Street address |
Rämistrasse 100
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| City |
Zürich |
| ZIP/Postal code |
8091 |
| Country |
Switzerland |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE179364 |
Deviated myeloid differentiation in SCD mice under heme stress II. |
| GSE179365 |
Heme-activated Nrf2 signaling skews fate trajectories of bone marrow cells from dendritic cells towards macrophages |
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| Relations |
| BioSample |
SAMN20013882 |
| SRA |
SRX11343040 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5416218_Spleen_positive_selection_Berkley_barcodes.tsv.gz |
74.4 Kb |
(ftp)(http) |
TSV |
| GSM5416218_Spleen_positive_selection_Berkley_features.tsv.gz |
225.2 Kb |
(ftp)(http) |
TSV |
| GSM5416218_Spleen_positive_selection_Berkley_matrix.mtx.gz |
103.3 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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