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65,000 proliferating erythroid cells from the buffy coat of a blood donation were obtained by flow cytometric separation after a 5-day culture period in the presence of erythropoietin. Total RNA was purified from the sorted cell population using TRIzol reagent. RNA (0.3 ug) was converted into double stranded cDNA using Clontech's CapFinder cDNA Library Construction Kit (Clontech) according to the manufacturer's protocol and cloned into EcoRI digested Lambda Zap II vector (Stratagene). The phage library was amplified once prior to in vivo excision in SOLR cells. Individual colonies were grown, and the cDNA inserts were sequenced in high throughput (NIH intramural sequencing center http://www.nisc.nih.gov/).
Nucleotide
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