Quantitative RT-PCR Analysis of PU.1 Expression

Fluidigm qRT-PCR analysis was performed as previously described [7 (link)]. Briefly, 100 PU.1 low and PU.1 high SLAM cells per well were sorted directly into 5 µL of 2× Reaction Mix (Invitrogen, Waltham, MA, USA) contained within a 96-well PCR plate. Once all cells were sorted, the plates were sealed with an aluminum seal, spun in a centrifuge at 1200 RPM for 5 min, snap frozen in liquid nitrogen, and stored at −80 °C. cDNA was generated from RNA using Superscript III (Invitrogen) and a custom primer set mix (Fluidigm, San Francisco, CA, USA) that were amplified for 18 cycles on a thermocycler (Eppendorf, Hamburg, Germany). Excess primers were removed from the samples by Exonuclease I (NEB, Ipswich, MA, USA) treatment, and the samples were diluted in DNA suspension buffer (Teknova, Hollister, CA, USA). Pre-amplified cDNA and custom primer sets were loaded onto a Fluidigm 96.96 Dynamic Gene Expression IFC. Subsequently, the IFC was run on a Biomark HD (Fluidigm) with SsoFast Sybr Green (Bio-Rad, Hercules, CA, USA) used for detection. Fluidigm gene expression software was used to analyze the data, and all values are relative to Gusb. Relative changes in gene expression were determined using the ΔΔCT approach.