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Sample GSM4116049 Query DataSets for GSM4116049
Status Public on Nov 25, 2019
Title CD34+ cord blood cells, d2_UM171_1000nM
Sample type SRA
Source name umbilical cord blood CD34+
Organism Homo sapiens
Characteristics cell type: CD34+ cord blood cells
ex vivo culture: 48h
treatment: UM171_1000nM
Treatment protocol CD34+ cultures were supplemented with either DMSO, 35 nM or 1000 nM UM171 (StemCell Technologies).
Growth protocol Human CD34+ cells were cultured in HSC expansion media consisting of StemSpan SFEM (StemCell Technologies) supplemented with human 100 ng/ml stem cell factor (SCF, R&D Systems), 100 ng/ml FMS-like trysine kinase 3 ligand (FLT3, R&D Systems), 50 ng/ml thrombopoietin (TPO, R&D Systems), and 10 μg/ml low-density lipoprotein (StemCell Technologies).
Extracted molecule total RNA
Extraction protocol Single cells were processed using the 10X Chromium controlller.
10X Chromium version 2 3' RNAseq.
Illumina NovaSeq 6000 S2
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Data processing CD34+ cord blood (CB) cells were cultured for 48h, a timepoint before most cells undergo division. Experimental conditions included either DMSO or two different UM171 concentrations; 35nM, a dose previously established as optimal for HSC expansion as well as 1000 nM. Single-cell RNAseq from each of these cultures was performed on a Chromium Single-Cell Controller (10X Genomics) using the Single Cell 3’ Reagent Kit version 2 according to manufacturer’s instructions. Target cell numbers were 6,000 per condition. Sequencing of the scRNAseq libraries was performed on an Illumina NovaSeq device using a S2 (PE 28x91) setup. A standard Cellranger v3.0.1 pipeline was used for read mapping (GRCh38 annotation) and demultiplexing. Subsequent analyses were done in Seurat (v2.3) and included (i) exclusion of cells with less than 1,000 genes or unique molecular identifiers (UMI), (ii) exclusion of cells with more genes or UMIs than the respective means plus 2 standard deviations (likely representing multiplets), (iii) exclusion of cells with more than 6% mitochondrial gene expression (representing apoptotic cells). Expression counts were log-normalized (scale factor 10,000) and scaled including regression on number of UMI’s, cell cycle scores and mitochondrial gene content. Multi-set Canonical Correlation Clustering and tSNE embedding was performed using variable genes (defined by mean.function = ExpMean, dispersion.function = LogVMR, x.low.cutoff = 0.02, x.high.cutoff = 5, y.cutoff = 2) excluding sex-specific genes.
Genome_build: GRCh38
Supplementary_files_format_and_content: Expression matrix where each row contains normalized gene expression obtained from after applying normalization and scaling using the Seurat v2.3 processing pipeline. Each column header contains the sample identifier and cell barcode identifier.
Submission date Oct 10, 2019
Last update date Nov 25, 2019
Contact name Guy Sauvageau
Organization name Institute for Research in Immunology and Cancer
Street address 2950, Chemin de Polytechnique
City Montreal
State/province QC
ZIP/Postal code H3T 1J4
Country Canada
Platform ID GPL24676
Series (1)
GSE138680 UM171 induces a homeostatic inflammatory-detoxification response supporting human HSC self-renewal
BioSample SAMN13008695
SRA SRX6974900

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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