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| Status |
Public on Mar 31, 2017 |
| Title |
5FU_Day10_22 |
| Sample type |
SRA |
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| Source name |
Bone Marrow
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| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6
cell type: GMP
treatment: 5-FU
time: Day10
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| Extracted molecule |
total RNA |
| Extraction protocol |
Six- to 12-weeks-old C57Bl/6 wilt type mice were treated with a single dose of 150 mg/kg 5-FU (Sigma-Aldrich) or with PBS control and were analyzed at different time points (0, 8, 10, 12 and 14 days) post-treatment. Bone marrow (BM) cells were obtained by either crushing leg, arm and pelvic bones or just flushing leg bones in Hanks’ Buffered Saline Solution (HBSS) containing 2% heat-inactivated FBS (Sigma-Aldrich). HBSS/2% FBS was used for all incubation and wash steps. Erythrocytes and contaminating bone material were removed by ACK lysis followed by centrifugation on a Ficoll gradient (Histopaque 1119, Sigma-Aldrich) for crushed bones. BM cells were first pre-enriched for c-Kit+ cells using c-Kit microbeads (Miltenyi Biotec, 130-091-224) and MACS Separation LS Columns (Miltenyi Biotec, 130-042-401), and incubated with purified, unconjugated-lineage antibodies (CD3 from Biolegend, and CD4, CD5, CD8, B220, Ter119, Mac-1 and Gr-1 from eBioscience) followed by goat anti-rat-PE-Cy5 (Invitrogen, A10691) and subsequently blocked with purified rat IgG (Sigma-Aldrich). Cells were then stained with c-Kit-APC-eFluor780 (eBioscience, 47-1171-82), Sca-1-PB (BioLegend, 108120), CD48-A647 (BioLegend, 103416), CD150-PE (BioLegend, 115904), CD34-FITC (eBioscience, 11-0341-85) and FcγR-PerCP-eFluor710 (eBioscience, 46-0161-82) after 5-FU treatment. Single GMPs (lin-/c-kit+/sca-1-/cd34+/fcgr+) were directly sorted into individual wells of a 96-well PCR plate containing 2.3 µl of 0.2% Triton-X100 (Sigma-Aldrich, Cat. no. 93443) and 2.3 U of SUPERase-In RNase Inhibitor (Ambion, Cat no. AM2694). cDNA was obtained and amplified following the described SMARTSEQ2 protocol (Picelli et al., 2014). Libraries were prepared for sequencing from 125 ng of cDNA using the Illumina Nextera XT DNA preparation kit. Pooled libraries were run on the Illumina Hi-Seq 2500.
Libraries were prepared for sequencing from 125 ng of cDNA using the Illumina Nextera XT DNA preparation kit.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
5FU_Day10_HTSeq_counts.txt.gz
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| Data processing |
RNA-seq reads were aligned to mm10 using GSNAP (version 2015-09-29) with parameters (-B 5 -t 24 -n 1 -Q -N 1).
Reads in features were counted with htseq-count (HTSeq version 0.5.3p3) with the parameter (-s no).
Genome_build: mm10
Supplementary_files_format_and_content: HT-Seq counts (.txt)
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| Submission date |
Nov 22, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Evangelia Diamanti |
| E-mail(s) |
ed347@cam.ac.uk
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| Phone |
01223 62317
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| Organization name |
University of Cambridge
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| Department |
Haematology
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| Lab |
Gottgens
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| Street address |
Wellcome Trust / MRC Building, Hills Road
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| City |
Cambridge |
| ZIP/Postal code |
CB2 0XY |
| Country |
United Kingdom |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE90140 |
Molecular reprogramming of granulocyte/macrophage progenitor (GMP) clusters upon 5-Fluorouracil (5-FU) treatment |
| GSE90799 |
Molecular reprogramming of granulocyte/macrophage progenitor (GMP) clusters |
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| Relations |
| BioSample |
SAMN06047524 |
| SRA |
SRX2367225 |
| 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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