Human UCB from full-term delivered infants was obtained after informed consent in accordance with guidelines approved by the University of Minnesota Committee on the Use of Human Subjects in Research. Each biologically-distinct replicate was comprised of one to four donors that were pooled prior to processing and microarray analysis. CD34+CD38-CD33-Rho(lo)c-kit+ and CD34+CD38-CD33-Rho(hi) fractions were selected by sequential Ficol-Hypaque separation, MACS column depletion and fluorescence activated cell sorting. Total cellular RNA was isolated using the PicoPure RNA Isolation Kit (Arcturus, Mountain View, California) per the manufacturer’s instructions. Seven to ten thousand Rho(lo) and Rho(hi) cells were sorted directly into 100 mcL Extraction Buffer (XB) provided with the PicoPure RNA Isolation Kit (Arctutus) prior to RNA isolation. Labeled complimentary-RNA (cRNA) was generated by one round of IVT-based, linear amplification using the RiboAmp OA RNA Amplification Kit (Arcturus) followed by labeling with the Enzo Bioarray™ HighYield™ RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, New York) according to manufacturer’s instructions. Affymetrix® HG-U133 GeneChips™ were processed using Genedata Refiner software (GeneData, San Francisco, California) to assess overall quality. Feature intensities for each chip were condensed into a single intensity value per gene using the Affymetrix Statistical Algorithm (MAS 5.0) with Tau = 0.015, Alpha1 = 0.04, Alpha2 0.06 and a scaling factor of 500.
