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Status |
Public on Dec 02, 2022 |
Title |
mWT_Antibody_Capture |
Sample type |
SRA |
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Source name |
mWT_Antibody_Capture
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Organism |
Mus musculus |
Characteristics |
tissue: Bone Marrow sample type: Antibody_Capture
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Extracted molecule |
other |
Extraction protocol |
BM was extracted from femurs, pelvic bones, tibias and spines of TAM-treated Nfixflox/floxRosa26CreERT2+/+ and Nfixflox/floxRosa26CreERT2+/T mice via crushing c-Kit+ cells were isolated via magnetic enrichment using anti-CD117 microbeads (Miltenyi Biotec, Carlsbad, CA) and an autoMACs magnetic cell separator (Miltenyi Biotec, Carlsbad, CA). For c-Kit+ cells, HSPC (Lineage-Sca-1+cKit+) and LT-HSC (Lineage-Sca-1+c-Kit+CD48-CD150+) isolation, cells were stained with anti-Lineage BV605 cocktail, anti-Sca-1-PerCPCy5.5 (E13-161.7), anti-c-Kit-APCe780 (2B8), anti-CD48-Alexa Fluor 700 (HM48-1), anti-CD150-PE-Cy7 (TC15-12F12.2). For CMP (Lineage-Sca-1-c-Kit+CD34+CD16/32med) isolation, cells were stained with anti-Lineage BV605 cocktail, anti-Sca-1-PerCP-Cy5.5 (E13-161.7), anti-c-Kit-APCe780 (2B8), anti-CD34-FITC (RAM34) and anti-CD16/32-Alexa Fluor 700 (2.4G2). In addition to FACS antibodies, cells were labeled with CITE-seq antibodies (see Supplementary Table S1). 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO) was used for dead cell exclusion. All cell isolations were performed using cell sorting on FACSAria III (BD Biosciences, San Jose, CA). Post cell sorting, 7000 Lineage-cKit+ BM, 1000 HSPC, 1000 CMP, and 1000 LT-HSC were pooled together for 10x genomics
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Library strategy |
RNA-Seq |
Library source |
other |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
CITE-seq manual_label.RData
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Data processing |
Single cell data was analyzed using cellranger count. Transcriptome database was cellranger-mm10-3.0.0. The weighted nearest-neighbor UMAP and cell clusters were generated using Seurat (v4.0.4) following the reference guide of “WNN analysis of CITE-seq, RNA+ADT” Differential gene expression analysis was done using FindMarkers with “slot=data, logfc.threshold=0.2, test.used=wilcox, pseudocount.use=1”. Genes were further filtered by FDR<=0.01, which was calculated using the Benjamini-Hochberg method in R. Genome_build: mm10 Supplementary_files_format_and_content:Rdata
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Submission date |
Apr 13, 2022 |
Last update date |
Dec 02, 2022 |
Contact name |
Shannon McKinney-Freeman |
E-mail(s) |
Shannon.McKinney-Freeman@STJUDE.ORG
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Organization name |
St. Jude Children’s Research Hospital
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Department |
Department of Hematology
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Street address |
262 Danny Thomas Place
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City |
Memphis |
State/province |
TN |
ZIP/Postal code |
38018 |
Country |
USA |
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Platform ID |
GPL24247 |
Series (1) |
GSE166922 |
A Nuclear factor I-X mediated regulatory network governs the balance of hematopoietic stem and progenitor cells during hematopoiesis |
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Relations |
BioSample |
SAMN27562384 |
SRA |
SRX14841653 |
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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