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| Status |
Public on Dec 20, 2023 |
| Title |
B cells from bone marrow, scRNA-seq, HTO |
| Sample type |
SRA |
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| Source name |
bone marrow
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| Organism |
Mus musculus |
| Characteristics |
cell type: B cells
tissue: bone marrow
strain: C57BL/6
Sex: female
age: 8 weeks
library type: HTO
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| Extracted molecule |
protein |
| Extraction protocol |
Blood was drawn from the heart shortly after euthanization and collected into PBS with 2 mM EDTA. Inguinal lymph nodes, spleen, thymus and femurs were dissected. Bone marrow was extracted using centrifugation (10000g, 4 °C, 1 min). Lymph nodes, spleens, and thymi were passed through 100-μm cell strainers, followed by centrifugation (400g, 4 °C, 10 min). Spleen, bone marrow and blood samples underwent erythrocyte lysis with BD Pharm Lyse (BD Biosciences). All samples were pre-enriched for B cells using CD19 microbeads (130-121-301, Miltenyi Biotec) and Magnetic Activated Cell Sorting. Organs from two mice were pooled. Single-cell suspensions were incubated with LIVE/DEAD fixable dye (Invitrogen) and mouse Fc Block in PBS for 15 min on ice. Cells were washed with fluorescence-activated cell sorting (FACS) buffer (2% FCS in PBS) and incubated with fluorochrome-conjugated antibodies (1:100) against surface markers for 30 min on ice. Additionally, samples were labeled with TotalSeq-C anti-mouse Hashtags 1-3 and 5-6 (M1/42; 39-F11, Biolegend, 1:100 for all antibodies; HTO) as to the manufacturer's instruction. 12.000 live cells per organ were sorted for CD3-CD11b-F4/80-NK1.1-CD19+ B cells on a FACSAria III (BD Biosciences) into an FCS-coated 96-well plate.
scRNA-seq (GEX), scBCR-seq (VDJ), and cell hashing libraries (HTO) were prepared using the 10x Chromium Single Cell 5' Solution (Chromium Next GEM Single Cell VDJ v1.1 with feature barcoding technology for cell surface protein, 10x Genomics). In brief, sorted cells were transferred to a Chromium Next Gem chip (10x Genomics), and partitioning was performed automatically by Chromium Controller (10x Genomics). Library preparation was performed according to the manufacturer's instructions. For quality control and quantification, a Bioanalyzer 2100 (Agilent Technologies) was used. Cells from individual organs were processed in separate GEM wells, so hashtag antibodies served only as additional control in this dataset (no demultiplexing).
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| Library strategy |
RNA-Seq |
| Library source |
other |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10x Genomics
Sample_03_BM_HTO
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| Data processing |
Cellranger-7.1.0 count pipeline, with the `include introns` option disabled, was used to process FASTQ raw data (GEX and HTO together for each sample), align reads to the mm10 mouse reference genome and perform gene counting. VDJ data was not processed for this study.
Cellranger output files were further processed using the velocyto-0.17.17 run10x pipeline to generate spliced/unspliced matrices (loom files).
Filtered feature-barcode matrices were analyzed using SCANPY-1.9.2. For quality control, cells with more than 15% mitochondrial gene counts were excluded as well as cells with more than 2% hemoglobin gene counts and cells with less than 5% ribosomal gene counts. Cells with less than 200 detected genes and cells with more than 1E4 counts per cell were also removed. Genes detected in less than 3 cells were excluded. 254 variable B cell receptor chain genes detected in the dataset were excluded using the regex `^Ig[hjkl].*[-v]`. Doublet exclusion was performed using scrublet. Gene counts were normalized to 1E4 total counts per cell and variance was stabilized by log1p transformation.
Loom files were analyzed using scvelo-0.2.5 and UniTVelo-0.2.5.2.
Assembly: mm10-2020-A
Supplementary files format and content: Filtered feature-barcode matrices as generated using cellranger-7.1.0 count pipeline.
Supplementary files format and content: Loom files as generated using velocyto-0.17.17 run10x pipeline.
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| Submission date |
Jun 05, 2023 |
| Last update date |
Dec 20, 2023 |
| Contact name |
Thomas Korn |
| E-mail(s) |
thomas.korn@tum.de, c.sie@tum.de
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| Organization name |
Technical University of Munich
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| Department |
Experimental Neuroimmunology
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| Street address |
Ismaninger Str. 22
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| City |
Munich |
| ZIP/Postal code |
81675 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE234188 |
Gene expression profile at single cell level of murine B cells from different compartments of naive young adult mice |
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| Relations |
| BioSample |
SAMN35624997 |
| SRA |
SRX20595574 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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