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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 03, 2020 |
| Title |
ATAC-seq-Spleen-Sca-neg-#2 |
| Sample type |
SRA |
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| Source name |
Spleen day 8 after lethal irradiation and transplantation of unfractionated bone marrow cells
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| Organism |
Mus musculus |
| Characteristics |
strain background: B6SJL
cell type: Splenic stress-BFU-E
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| Extracted molecule |
genomic DNA |
| 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)
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Spleen-idr-ATAC-peaks-annotated-mm10.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).
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| 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 (https://usegalaxy.org/). Further analyses were performed using the HOMER platform (v4.8, v4.10)(Heinz et al. 2010). Expression matrixes were created using analyzeRepeats.pl 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 getDiffExpression.pl 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 (2.3.4.2) (Langmead, B. etal. 2009 and 2012). Peak calling was performed using the ENCODE ATAC-seq pipeline (https://github.com/kundajelab/atac_dnase_pipelines) (-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.
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| Submission date |
Oct 24, 2019 |
| Last update date |
Jan 03, 2020 |
| Contact name |
Johan Flygare |
| E-mail(s) |
johan.flygare@med.lu.se
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| Organization name |
Lund University
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| Lab |
Flygare
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| Street address |
Sölvegatan 19
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| City |
Lund |
| ZIP/Postal code |
22184 |
| Country |
Sweden |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE139336 |
Prospective isolation of radiation induced erythroid stress progenitors reveals unique transcriptomic and epigenetic signatures enabling increased erythroid output |
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| Relations |
| BioSample |
SAMN13108510 |
| SRA |
SRX7051466 |
| 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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