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| Status |
Public on Jan 31, 2024 |
| Title |
BMSCs, WT2 |
| Sample type |
SRA |
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| Source name |
Bone Marrow
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| Organism |
Mus musculus |
| Characteristics |
tissue: Bone Marrow
cell line: NA
cell type: Primary cells
genotype: WT
treatment: cell culture
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| Treatment protocol |
NA
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| Growth protocol |
Cells were cultured for 21 days in αMEM medium supplemented with 10% FBS (Corning, New York, USA), 10 U/ml penicillin, 100 μg/ml streptomycin (Thermo Fisher Scientific, Whaltman, USA), 10 mM β-glycerophosphate, and 50 μg/ml ascorbic acid (Sigma-Aldrich, St. Louis, USA) to induce differentiation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Collected cells were isolated after 5 min Trypsin 1X incubation. Cells were incubated with 7-Aminoactinomycin D (7AAD) for 10 minutes and kept on ice until fluorescence sorting. Live cells (7AAD negative) were filtered through a 40 µm nylon mesh, and isolated cells were loaded into the 10X Chromium system and libraries were constructed using Chromium Single Cell 3’ Reagent Kit (v3) according to the manufacturer’s protocol. Libraries were subsequently sequenced on a NovaSeq 6000 Sequencing System (Illumina).
Samples were multiplexed using the CellPlex kit (1 separate reaction per genotype) (10X Genomics).The sequencing data were demultiplexed using the Cell Ranger 7.0.1 pipeline (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 |
10X Genomics
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| Data processing |
After the initial demultiplexing we used Seurat HTODemux function to separate the data in 12 separate samples. Using the barcodes for each sample, data was demultiplexed in individual samples (bam, matrix, features and barcodes) using the 10 Genomics CellRanger with barcode-sample-assignment attribute. Alignment was performed against the mm10-2020-A build using both exonic and intronic reads.
Data from each individual sample was processed into a Seurat Object and mitochondiral percentage was calculated. Data from each sample was merged into a single Seurat Object, normalized using SCT transform and mitochondrial percentage was regressed.
Seurat clusters were called using 50 npcs, 1:30 umap reduction on the SCT assay and FindClusters function at the resolution of 0.8. Celltypes were detemined using differentially expressed gene markers in clusters.
An osteoblast subcluster was created from the intitial Seurat object and used for to determine the genes differentially expressed in osteoblastic clusters.
Assembly: mm10
Supplementary files format and content: Tab separated matrix, features and barcodes(cells) files.
Supplementary files format and content: FGF23 regulated and DMP1 regulated differential expression with corrected p value (BD FDR p<0.1).
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| Submission date |
Aug 01, 2023 |
| Last update date |
Jan 31, 2024 |
| Contact name |
Valentin David |
| E-mail(s) |
valentin.david@northwestern.edu
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| Phone |
13125034159
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| Organization name |
Northwestern University
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| Department |
Medicine
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| Lab |
David Lab
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| Street address |
320 E Superior Street
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| City |
Chicago |
| State/province |
Illinois |
| ZIP/Postal code |
60611 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE239828 |
Role of FGF23 and DMP1 in osteoblastogenesis in cultured BMSCs at single cell level |
| GSE239830 |
Role of FGF23 and DMP1 in osteoblastogenesis |
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| Relations |
| BioSample |
SAMN36792820 |
| SRA |
SRX21215753 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7674899_WT2_barcodes.tsv.gz |
10.9 Kb |
(ftp)(http) |
TSV |
| GSM7674899_WT2_features.tsv.gz |
284.2 Kb |
(ftp)(http) |
TSV |
| GSM7674899_WT2_matrix.mtx.gz |
18.9 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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