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Sample GSM7453699 Query DataSets for GSM7453699
Status Public on Dec 20, 2023
Title B cells from bone marrow, scRNA-seq, HTO
Sample type SRA
Source name bone marrow
Organism Mus musculus
Characteristics cell type: B cells
tissue: bone marrow
strain: C57BL/6
Sex: female
age: 8 weeks
library type: HTO
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).
Library strategy RNA-Seq
Library source other
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Description 10x Genomics
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-
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.
Submission date Jun 05, 2023
Last update date Dec 20, 2023
Contact name Thomas Korn
Organization name Technical University of Munich
Department Experimental Neuroimmunology
Street address Ismaninger Str. 22
City Munich
ZIP/Postal code 81675
Country Germany
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
BioSample SAMN35624997
SRA SRX20595574

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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