Cells were stained and sorted using anti–CD36-PE (BD Biosciences; used for CFU-Es, Pro-Es and Int-Es), anti–CD71-FITC (DakoCytomation), anti–CD235a-APC (BD Biosciences; used for CFU-Es, Pro-Es and Int-Es), and anti–CD235a-RPE (DakoCytomation; used for Late-Es).
Growth protocol
Human primary differentiating erythroblasts derived from peripheral blood buffy coat (NHSBT, Bristol, UK) were obtained with informed consent in accordance with the declaration of Helsinki. Erythroblasts were cultured using a two-phase liquid culture system as previously described. In brief, PBMCs were obtained from peripheral blood buffy coat by Ficoll-Hypaque density-gradient centrifugation and seeded at 2.5 x 106 cells/ml in MEM with 10% FCS, 1 µg/ml cyclosporin A, and 10% conditioned medium from the 5637 bladder carcinoma cell line. The cells were cultured for 6–7 d (phase I), and the non-adherent cells were washed and re-seeded in fresh medium with 30% FCS, 1% deionized BSA, 1 U/ml of human recombinant erythropoietin (EPO), 10-5 M β-mercaptoethanol, 10-6 mol/l dexamethasone, 0.3 mg/ml holotransferrin (ICN Biochemicals), and 10 ng/ml of human stem cell factor (phase II). Maturation of erythroblasts was monitored by May Grünwald-Giemsa stained cytospin preparations using light microscopy. Cells were harvested following phase II culture in the presence of EPO for four, five-six, eight-eleven or thirteen-fifteen days to obtain populations of CFU-Es, Pro-Es, Int-Es, or Late-Es, respectively.
Extracted molecule
total RNA
Extraction protocol
Following confirmation of purity of the sorted erythroblast population by examining cell morphology on cytospins, total RNA was extracted using Trizol (Invitrogen, Paisley, UK) in accordance with the manufacturer’s instructions with an additional phenol-chloroform step. RNA integrity was evaluated using an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
Label
biotin
Label protocol
For each of the four stages of erythroid precursors included in the study, three RNA pools were prepared from non-overlapping sets of four different RNA preparations mixed in equal proportions. One RNA pool of four RNA preparations from unsorted cells harvested at each of the four different stages of culture was also prepared for comparison. Biotinylated fragmented cRNA was synthesized from 100ng of pooled RNA using the 2-Cycle cDNA Synthesis and the 2-Cycle Target Labelling and Control Reagent kits (Affymetrix, Santa Clara, CA), and hybridized to GeneChip Human Genome U133_Plus_2.0 arrays (Affymetrix) following the manufacturer’s recommendations.
Hybridization protocol
Biotinylated cRNA was hybridized to GeneChip Human Genome U133_Plus_2.0 arrays (Affymetrix) following the manufacturer’s recommendations. Briefly, hybridisation was for 16 hours at 45 degrees C, rotating at 60rpm.
Scan protocol
Genechips were scanned using an Affymetrix GeneChip scanner following washing on a Fluidics Station 450.
Description
RNA was pooled from 4 samples of Pro-E stage PBMC-derived sorted erythroblasts.
Data processing
Cell intensity calculation and scaling was performed using GeneChip Operating Software (GCOS) (Affymetrix) and data analysed by the Robust Multiarray Average (RMA) method of Irrizarry using the Bioconductor suite of packages in R (www.bioconductor.org).
