characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2014-10-08T17:01:00Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:49:48Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2223988
characteristics.characteristic: External Id
characteristics.text: EMBL
characteristics.characteristic: INSDC center name
characteristics.text: 2014-10-08T17:01:00Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T16:49:48Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS360416
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-1963:CELLS_LS-K1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: myeloid comitted progenitor cells
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Bone marrow was isolated from hindlegs (femur, tibia), hips (ilium) and backbone (vertebra). Muscle, connective tissue and spinal cord were removed, bones were crushed in RPMI/ 2 percent FBS (Gibco, Carlsbad, CA) using mortar and pestle. Single cell-suspensions were made by flushing through a 40 um filter mesh. Cell numbers and viability were determined using a ViCell Counter (Beckman Coulter, Brea, CA). To deplete lineage-positive cells, total bone marrow was stained 30 minutes with a combination of the following monoclonal rat antibodies directed against mature cell specific lineage markers: anti-CD4 (clone GK1.5), anti-CD8a (53.6.7), anti-CD11b (M1/70), anti-B220 (RA3.6B2), anti-GR1 (RB6.8C5) and anti-TER119 (Ter119). Labeled cells were incubated for 20 minutes with polyclonal sheep anti-rat IgG coated magnetic Dynabeads (Invitrogen, Hercules, CA) at a ratio 2:1 beads to cell and depleted using a magnet, enriching for the lineage-negative (Linneg) cell fraction. Beads were washed twice with RPMI/ 2 percent FBS to harvest residual cell fractions. Centrifugation steps were carried out at 1,500 rpm and 4 degrees C for 5 minutes (5810r, Eppendorf, Hamburg, Germany). To specify multipotent and myeloid progenitor fractions, the Linneg fraction was stained 30 minutes using the following rat monoclonal fluorochrome-coupled antibodies: anti-CD4 (GK1.5), anti-CD8a (53.6.7), anti-CD11b (M1/70), anti-B220 (RA3-6B2), anti-Gr1 (RB6.8C5) and anti-Ter119, all phycoerythrobilin-cyanine dye 7 (PE-Cy7) conjugated; anti-CD117/c-Kit (2B8)- PE; anti-Sca-1 (D7)-Alexa700. Lineage antibodies for depletion of mature effector cells were purified and conjugated in our laboratory according to standard protocols. Monoclonal antibody conjugates were purchased from eBioscience (San Diego, CA) or BioLegend (San Diego, CA). All antibodies were titrated prior to use. Cell sorting was performed on a FACS Aria I or FACS Aria II (Becton Dickinson, San Jose, CA) at the DKFZ Flow Cytometry Service Unit, Heidelberg, Germany. The following sort parameters were used: 70 um nozzle; 15,000 evt/s.; 70 psi. Multipotent hematopoietic stem/progenitor cells (LS+K) and myeloid comitted progenitor cells (LS-K) were obtained by sorting LinnegSca-1+c-Kit+ and LinnegSca-1+c-Kit-, respectively, in biological triplicate. Sorted cells were collected into ice-cold RNA lysis buffer (ARCTURUS(R) PicoPure(R) RNA Isolation Kit (Life Technologies, Invitrogen)) and stored at -80 degrees C until further usage. Of note, to determine sample's purity we re-sorted a fraction of each population (each population showed a purity greater than 95 percent). Total RNA isolation was performed from the indicated populations using ARCTURUS(R) PicoPure(R) RNA Isolation Kit (Life Technologies, Invitrogen) according to the manufacturer's instructions. DNAse treatment was performed using RNAse-free DNAse Set (Qiagen). Total RNA was used for quality controls and for normalization of starting material. cDNA-libraries were generated with 10 ng of total RNA using the SMARTer(TM) Ultra Low RNA Kit for Illumina Sequencing (Clontech) according to the manufacturer's indications. Of note, twelve cycles were used for the amplification of cDNA respectively. Paired-end adaptors were applied to each population. Sequencing was performed with the HiSeq2000 device (Illumina) and one sample per lane.
characteristics.characteristic: description
characteristics.text: adult early hematopoiesis
characteristics.characteristic: developmental stage
characteristics.text: wild type
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005168
characteristics.characteristic: genotype
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: fresh specimen
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000971
characteristics.characteristic: specimen with known storage state
characteristics.text: C57BL/6J
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0000606
characteristics.characteristic: strain
characteristics.text: CELLS_LS-K1
characteristics.characteristic: title