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Sample GSM4138362 Query DataSets for GSM4138362
Status Public on Jan 03, 2020
Title RNA-seq-Spleen-Sca-pos-#2
Sample type SRA
Source name Spleen day 8 after lethal irradiation and transplantation of unfractionated bone marrow cells
Organism Mus musculus
Characteristics strain background: B6SJL
cell type: Splenic stress-MPP
Extracted molecule total RNA
Extraction protocol For ATAC-seq: sorted cells were washed in ice cold PBS prior to Assay for Transposase Accessible Chromatin (ATAC-seq) library preparation was performed essentially as described by Buenrostro et al. 2013, using Transposomes from Nextera DNA library preparation kit (Illumina, San Diego, CA) and custom primers. Sequencing was performed on a NextSeq500 (Illumina, San Diego, CA, USA). RNA-seq libraries were constructed using SMARTer Stranded Total RNA-Seq Kit v2 (Pico Input Mammalian; Takara Bio USA, Mountain View, CA, USA) followed by sequencing on a HiSeq3000 (Illumina, San Diego, CA, USA)
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 3000
Description AnalyzeRepeats-Spleen-samples-Sca-neg_Sca-pos_CD9-neg_count-exons_condeseGenes_noadj.txt
Bone marrows (BM) and spleens were homogenized and filtered to single cell suspensions. Prior to FACS, enrichment for progenitors was carried out by lineage depletion using unconjugated antibodies produced in rat against murine B220, CD4, CD8, Gr-1 and Ter-119 (all from BioLegend, San Diego, CA, USA). Lineage-positive cells were removed with a magnetic particle concentrator (MPC-6; ThermoFisher Scientific, Göteborg, Sweden) and sheep anti-rat Fc-conjugated immunomagnetic beads (ThermoFisher Scientific).
For FACS, the following antibodies against murine epitopes were used: CD71 (FITC; BioLegend or biotin; BD Biosciences), CD9 (PE or FITC, clone MZ3; BioLegend), CD24a (PE-Cy7, clone M1/69; BioLegend), CD150 (APC, clone TC15-12F12.2; BioLegend), CD133 (PE, clone 315-2C11; BioLegend), c-Kit (APC780; eBioscience/ThermoFisher Scientific), Sca1 (BV421; BioLegend), B220 (Biotin or PE-Cy5; BioLegend), CD3 (Biotin or PE-Cy5; BD Biosciences), Gr-1 (Biotin or PE-Cy5; BioLegend) and Ter-119 (Biotin or PE-Cy5; BioLegend). All biotinylated antibodies were detected with Streptavidin-conjugated Qdot605 (ThermoFisher Scientific), except for the sorting of steady-state progenitors, where Qdot655 (Life Technologies/ThermoFisher Scientific, Göteborg, Sweden) was used instead. Fc receptor block (BD Biosciences) was used for sorting samples, and dead cells were excluded using 7-aminoactinomycin D (7-AAD; Sigma-Aldrich Sweden, Stockholm, Sweden) or 4',6-diamidino-2-phenylindole (DAPI; ThermoFisher Scientific). Fluorescence activated cell sorting (FACS) was performed on BD FACS Aria IIu or Aria III (Becton Dickinson).
Data processing RNA-Sequencing and data analysis: Biological samples were processed and sequenced in triplicate. For analysis of RNA-Seq experiments the reads were aligned to mouse reference genome (mm10 / GRCm38) using STAR STAR (2.6.0b-1) (Dobin, A etal. 2012),) with single read, standard settings on the Galaxy platform ( Further analyses were performed using the HOMER platform (v4.8, v4.10)(Heinz et al. 2010). Expression matrixes were created using with settings mm10 rna and options -count exons, -condeseGenes and -rpkm for normalized expression or -noadj for files for downstream statistical analyses. Statistically different expressed genes were identified using the created raw expression matrix with genes, with less than a mean of 50 raw tags in at least one sample filtered out, and the command using DESeq2 (REF) as statistical tool.
ATAC-seq: Biological samples were processed and sequenced in triplicate. For analysis of ATAC-Seq experiments the reads were aligned to mouse reference genome (mm10 / GRCm38) using Bowtie2 ( (Langmead, B. etal. 2009 and 2012). Peak calling was performed using the ENCODE ATAC-seq pipeline ( (-species mm10, -enable_idr, -no_xcor). Optimal IDR peaks were used for downstream analyses. Further analyses were performed using the HOMER platform (v4.8, v4.10) (Heinz et al. 2010).
Genome_build: mm10
Supplementary_files_format_and_content: Processed files include txt files of RNA-seq expressionmatrix and annotated-ATAC-peaks. All genomic coordinates are relative to mm10 mouse assembly.
Submission date Oct 24, 2019
Last update date Jan 03, 2020
Contact name Johan Flygare
Organization name Lund University
Lab Flygare
Street address Sölvegatan 19
City Lund
ZIP/Postal code 22184
Country Sweden
Platform ID GPL21493
Series (1)
GSE139336 Prospective isolation of radiation induced erythroid stress progenitors reveals unique transcriptomic and epigenetic signatures enabling increased erythroid output
BioSample SAMN13108495
SRA SRX7051481

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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