Genome wide H3K27ac status of endometrial stromal cells stimulated by cAMP, and cAMP under C/EBPb-knockdown

To know the role of C/EBPb in the changes of H3K27ac during desidualization, we compared the H3K27 profiles between cAMP-stimulation and cAMP-stimulation under C/EBPb-knockdown. Overall design: ESCs were transfected with control or C/EBPb siRNA and then treated with or without cAMP for 4 days. Cells cultured simultaneously with RNA-sequence experiment were cross-linked by adding formaldehyde to the medium at a final concentration of 1% and incubated for 10 min at 37 °C. Cross-linking was terminated by addition of glycine (0.125 M, final concentration). ChIP was performed with H3K27ac antibody. An unimmunoprecipitated control and immunoprecipitated DNA were used to prepare the ChIP-seq library with NEBNext DNA sample prep master mix set 1 (New England BioLabs, Ipswich, MA) and DNA sample prep kit, oligo only (Illumina) according to the manufacturer's instructions. After the quality of the library was validated using the Agilent Bioanalyzer 2100 (Agilent Technology), the ChIP-sequence library was sequenced by the Genome Analyzer GAIIx. The obtained reads from the sequencing platform was first applied for quality checking by the fastx quality stats program in the FASTX-Toolkit package (http://hannonlab.cshl.edu/fastx_toolkit/). The reads that passed the quality checking were then mapped to the human reference genome sequence (hg19) by using the bowtie program followed by Model-Based Analysis of ChIP-Sequence (MACS) to detect the H3K27ac regions.