organism: Mus musculus
description: Protocols: Bone marrow cells were isolated by flushing femurs, tibias, ilia and humerus with PBS. Cells were filtered through a 70µM cell strainer to obtain single cell suspensions and cell number was assessed by counting with a hemocytometer. Cell suspensions were first enriched for lineage-negative cells by MACS using Mouse Lineage cell depletion kit (Miltenyi Biotec) before staining for cell-surface antigens. Cells were stained for 30 minutes using the following rat anti-mouse monoclonal antibodies: Lineage cocktail (145-2C11, RA3-6B2, RB6-8C5, M1/70 and TER-119)-PE, CD34 (RAM34) -FITC, CD150 (Q8-480)-BV421, CD150 (mShad150) - PerCP-eFluor710, CD48 (HM48-1) -APC-Cy7, CD127 (SB/199) -V450. All reagents were purchased from ThermoFisher Scientific, BD Bioscience or BioLegend. For RNA-sequencing of Myeloid Progenitors (MyPs), 90000 MyPs (Lineage- CD127- c-kit+ Sca1- cells) were FACS-sorted on a FACS Aria 2 (BD Bioscience). MyPs were directly sorted into 1 ml of TRIzol Reagent (ThermoFisher Scientific). RNA was extracted using Direct-zol™ RNA MiniPrep Plus (Zymo Research) and cleaned of potential DNA contamination with RNeasy MinElute Cleanup Kit (Qiagen). LT-HSCs were directly FACS-sorted in 2 µl of Reaction Buffer from SMART-SeqTM v4 UltraTM Low input RNA Kit. For MyPs, cDNA was synthesized with 10 ng of total RNA using the SMART-SeqTM v4 UltraTM Low input RNA Kit for Sequencing (Clontech Laboratories) and amplified by 7 cycles of LD PCR. For LT-HSCs, cDNA was generated directly and amplified through 14 cycles using the SMART-SeqTM v4 UltraTM Low input RNA Kit for Sequencing (Clontech Laboratories).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-07-26T04:02:42Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-07-05T10:40:03Z
attributes.tag: External Id
attributes.value: SAMEA4777680
attributes.tag: INSDC center name
attributes.value: Laboratory of Cellular and Molecular Immunology GIGA-Research Center, Liege University
attributes.tag: INSDC first public
attributes.value: 2019-07-26T04:02:42Z
attributes.tag: INSDC last update
attributes.value: 2018-07-05T10:40:03Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6992:Elp3HscKO Myeloid Progenitors (rep3)
attributes.tag: age
attributes.value: 8 to 12
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: myeloid lineage restricted progenitor cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: genotype
attributes.value: Elp3fl/fl vav-iCreT/+
attributes.tag: organism part
attributes.value: bone marrow
attributes.tag: phenotype
attributes.value: CD127- c-kit+ Sca1-
attributes.tag: sample name
attributes.value: E-MTAB-6992:Elp3HscKO Myeloid Progenitors (rep3)
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: mixed pool of male and female
attributes.tag: strain
attributes.value: C57BL/6