characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2017-05-04T17:01:25Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-05-16T18:32:20Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104016725
characteristics.characteristic: External Id
characteristics.text: unspecified
characteristics.characteristic: INSDC center name
characteristics.text: 2017-05-04T17:01:25Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-05-16T18:32:20Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1681563
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-4547_2:CELL_dHSC_34
characteristics.characteristic: Submitter Id
characteristics.text: 150 days chasing
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: dormant hematopoietic stem cell
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% FBS (Gibco, Carlsbad, CA) using mortar and pestle. Single cell-suspensions were made by flushing through a 40 µm 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% FBS to harvest residual cell fractions. Centrifugation steps were carried out at 1,500 rpm and 4°C for 5 minutes (5810r, Eppendorf, Hamburg, Germany). To specify hematopoietic stem and progenitor cells, the Linneg fraction was stained 30 minutes at fixed cell density using the following rat monoclonal fluorochrome-coupled antibodies: anti-Streptavidin, phycoerythrobilin-cyanine dye 7 (PE-Cy7) conjugated; anti-CD117/c-Kit (2B8)-APC-eFl780; anti-Ly6a/Sca-1 (E13-161.7)-APC; anti-CD34 (RAM34)-FITC; anti-CD135 (A2F10)-PE; anti-CD150 (TC15-12F12.2)-PE-Cy5; anti-CD48 (HM-48-1)-PB. 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 µm nozzle; 10,000 evt/s.; 70 psi. dHSc, aHSC and MPP1 by sorting as described (Wilson et al. 2008), respectively, in biological triplicate. Sorted cells were collected into PBS. Of note, to determine sample’s purity we re-sorted a fraction of each population (each population showed a purity greater than 95%). Reverse transcription and amplification of cDNA was performed on-chip using the Smarter Ultra Low RNA Kit (ClonTech Cat#634834) chemistry according to Fluidigm C1 protocol.
characteristics.characteristic: description
characteristics.text: Adult early hematopoiesis
characteristics.characteristic: developmental stage
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: Bone marrow
characteristics.characteristic: organism part
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: SCLtTA;H2bGFP
characteristics.characteristic: strain
characteristics.text: CELL_dHSC_34
characteristics.characteristic: title