Growth Protocol

CD34+ cells, isolated from the peripheral blood of granulocyte colony-stimulating factor mobilized healthy volunteers, were obtained from the Yale Center of Excellence in Molecular Hematology. The cells were maintained and differentiated as previously described (Sankaran et al., 2008). Briefly the cells were expanded in StemSpan medium (Stem Cell Technologies Inc.) supplemented with 1X CC100 cytokine mix (Stem Cell Technologies Inc.) and 2% P/S for a total of 6 days. For studying the binding in differentiated cells after day 6 of expansion, cells were reseeded in differentiation medium (StemSpan SFEM Medium with 2% P/S, 20 ng/ml SCF, 1 U/ml Epo, 5 ng/ml IL-3, 2 uM dexamethasone, and 1 uM β-estradiol), at a density of 0.5-1 X 106 cells/ml till harvesting. At day 5 of differentiation cells reached the proerythroblast stage of erythroid differentiation.

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Treatment Protocol

At day 5 of differentiation cells reached the proerythroblast cells were stimulated for 2hrs with 5µM BIO (GSK3 inhibitor IX - Calbiochem 361550) and harvested for performing chromatin immunoprecipitation.

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Extraction Protocol

Whole cell extracts were sonicated to solubilize the chromatin. The chromatin extracts containing DNA fragments with an average size of 1000 bp were immunoprecipitated using different antibodies. Purified immunoprecipitated DNA were prepared for sequencing according to a modified version of the Solexa Genomic DNA protocol. Fragmented DNA was end repaired and subjected to 18 cycles of LM-PCR using oligos provided by Illumina. Amplified fragments between 150 and 300bp (representing shear fragments between 50 and 200nt in length and ~100bp of primer sequence) were isolated by agarose gel electrophoresis and purified.

Name

WCE_CD34prog_bmp_r1_101201_7

Organism

Homo sapiens

Cell type

Hematopoietic stem cell (HSC)

Disease state

Platform

Illumina Genome Analyzer II