NextSeq 500 paired end sequencing; RNA-seq of long-term hematopoietic stem cells and myeloid progenitors of control mice or mice conditionally deficient for Trp53 or Elp3 or both genes in the hematopoietic system
RNA-seq of long-term hematopoietic stem cells and myeloid progenitors of control mice or mice conditionally deficient for Trp53 or Elp3 or both genes in the hematopoietic system
Trp53HscKO Myeloid Progenitors (rep3)_p
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).