GSM2399522: 5FU_Day8_Rep1_23; Mus musculus; RNA-Seq
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.
GEO
GSM2399522
GEO Accession
GSM2399522