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| Status |
Public on May 01, 2024 |
| Title |
bone marrow, males from uninfected mothers, rep1, V(D)J-derived cDNA |
| Sample type |
SRA |
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| Source name |
bone marrow
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| Organism |
Mus musculus |
| Characteristics |
tissue: bone marrow
cell type: Immature and T1 B cells
genotype: 4getKN2
treatment: Born to uninfected (control) mothers
Sex: Male
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| Extracted molecule |
total RNA |
| Extraction protocol |
Femurs were harvested from pups either born to S. mansoni infected mothers or uninfected mothers at 28-35 days old. The end of the bone connected to the knee is cut, exposing bone marrow. Using a needle, a hole was punched into the bottom of a 200ul tube, and the bone is placed inside, cut side down, with 50ul of media. This tube was then placed in a 1.5mL tube and then spun at <10,000 RPM for 15 seconds at 4 degrees. Excess media was discarded, and cells were lysed with BD lysis buffer diluted to 1X for 30 seconds. The reaction was quenched using 1%FBS and cells were pelleted by centrifugation at 200xg for 5 minutes at 4 degrees. Primary antibody staining was done for 30 minutes in the dark at 4 degrees and washed twice before being filtered through a 40um filter and pooled. At least 3 mice were used per pool. Then the cells were sorted on a FACS Aria. After sorting, cells were stained with TotalSeq antibodies per manufacture’s instruction. Cells were counted and resuspended at a concentration of 10,000 cells/ul before proceeding to library construction.
Library was prepared using Chromium Single Cell 5ʹ v2 protocol (10x Genomics). Cellular mRNA and antibody-derived oligos were reverse-transcribed and amplified by PCR according to the Chromium Single Cell 5’ v2 protocol (10x Genomics) and with specific primers amplifying the antibody-derived tags. For all cDNA library preparations, we further proceeded according to the manufacturer’s instructions for Single Cell 5’ v2 protocol (10x Genomics). mRNA and ADT sequences were combined in Cell Ranger, outputting one file per sample for those two libraries.
V(D)J library: Library was prepared using Chromium Single Cell 5ʹ v2 protocol (10x Genomics)
CITEseq library: Library was prepared using Chromium Single Cell 5ʹ v2 protocol (10x Genomics)
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
V(D)J/BCR
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| Data processing |
Pre-processing of sequencing results to generate count matrices (gene expression, V(D)J and ADT) was performed using the 10x genomics Cell Ranger pipeline where ADT and V(D)J sequences were treated as Custom library with default settings (v5.0.0) and aligning to the mm10 genome build and Ensembl 93 annotations.
Further processing was done with Seurat v4.1.0 (cell and gene filtering, clustering, differential gene expression analysis) for mRNA and ADT. V(D)J was processed using scRepertoire.
Assembly: mm10-3.0.0
Supplementary files format and content: 10x Genomics output files:filtered_feature_bc_matrix.h5
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| Submission date |
May 06, 2022 |
| Last update date |
May 01, 2024 |
| Contact name |
Keke Celeste Fairfax |
| E-mail(s) |
keke.fairfax@path.utah.edu
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| Phone |
810-581-5980
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| Organization name |
University of Utah
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| Department |
Pathology
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| Street address |
15 N Medical Drive East
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| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84112 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE202403 |
Maternal Schistosomiasis Bone Marrow B Cell V(D)J scRNAseq |
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| Relations |
| BioSample |
SAMN28117287 |
| SRA |
SRX15195040 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6118830_UM_filtered_contig_annotations.csv.gz |
984.4 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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