Identification of Hematopoietic Cell Markers

Cell surface markers were identified with the following fluorochromes: human hematopoietic lineage markers (CD2, CD3, CD14, CD16, CD19, CD56, CD235a) conjugated to APC (eBioscience), CD34 PE, CD38 Alexa Fluor 700, CD49f BV421, and CD45RA Brilliant Violet 570 (all from BioLegend). Appropriate isotype controls (BioLegend) were used for gating HSCs as described [29 (link), 30 (link)]. In some cases, following surface marker staining, cells were fixed with fixation buffer (BD Biosciences), permeabilized with Transcription Factor Fixation/Permeabilization Buffer kit (eBioscience) and stained with goat anti-human ARID3a, followed by rabbit anti-goat FITC secondary antibody (Invitrogen) to identify ARID3a-expressing cells and/or with H2A.X phopspho (Ser139) APC-Fire750 (Biologend). Human anti-ARID3a peptide specific antibodies were generated and isolated over a peptide-specific column as described [55 (link)]. Cells were pretreated with anti-human CD32 antibody to block Fc receptor binding prior to surface staining to exclude non-specific binding. Doublet exclusion was used to ensure analyses of single cells prior to forward/side scatter gating. Data were collected using an LSRII (BD Biogenics) and FACSDiva (BD Biosciences) software version 4.1 or Stratedigm S1200Ex and CellCapTure acquisition software and were analyzed using FlowJo (Tree Star) software version 10.