genotype: Wild type strain: Mixed 129/Svj and C57BL/6 tissue: Embryonic heart developmental stage: E17.5 chip antibody: H3K27me3
Extracted molecule
genomic DNA
Extraction protocol
20 embryonic hearts were fixed with 1% formaldehyde and lysed in lysis buffer (50mM Tris pH8.0, 10mM EDTA, 1% SDS, protease inhibitors). The crosslinked chromatin was sheared by sonication, and chromatin was diluted in dilution buffer (20mM Tris pH8.0, 2mM EDTA, 150mM NaCl, 1% Triton, 0.01% SDS, protease inhibitors). The samples were incubated with protein A agarose beads and H3K27me3 antibody ( (EMD Millipore, 07-449). The beads were washed with the following buffers; Wash buffer 1 (20mM Tris pH8.0, 2mM EDTA, 50mM NaCl, 1% triton, 0.1% SDS), Wash buffer 2 (10mM Tris pH8.0, 1mM EDTA, 250mM LiCl, 1% NP40, 1% Deoxycholic acid), TE buffer (10mM Tris pH8.0, 1mM EDTA). The protein-DNA complexes were eluted with elution buffer (100mM NaHCO3, 1% SDS) followed by the addition of proteinase K. Genomic DNA was isolated by phenol extraction and precipitation.
Label
Cy3
Label protocol
ChIP DNA was labeled with fluorescence per manufacturer's protocol
genotype: Wild type strain: Mixed 129/Svj and C57BL/6 tissue: Embryonic heart developmental stage: E17.5 chip antibody: none, input DNA
Extracted molecule
genomic DNA
Extraction protocol
20 embryonic hearts were fixed with 1% formaldehyde and lysed in lysis buffer (50mM Tris pH8.0, 10mM EDTA, 1% SDS, protease inhibitors). The crosslinked chromatin was sheared by sonication, and chromatin was diluted in dilution buffer (20mM Tris pH8.0, 2mM EDTA, 150mM NaCl, 1% Triton, 0.01% SDS, protease inhibitors). The samples were incubated with protein A agarose beads and H3K27me3 antibody ( (EMD Millipore, 07-449). The beads were washed with the following buffers; Wash buffer 1 (20mM Tris pH8.0, 2mM EDTA, 50mM NaCl, 1% triton, 0.1% SDS), Wash buffer 2 (10mM Tris pH8.0, 1mM EDTA, 250mM LiCl, 1% NP40, 1% Deoxycholic acid), TE buffer (10mM Tris pH8.0, 1mM EDTA). The protein-DNA complexes were eluted with elution buffer (100mM NaHCO3, 1% SDS) followed by the addition of proteinase K. Genomic DNA was isolated by phenol extraction and precipitation.
Label
Cy3
Label protocol
ChIP DNA was labeled with fluorescence per manufacturer's protocol
Hybridization protocol
Hybridized onto the Roche NimbleGen 3X720K RefSeq promoter arrays
Scan protocol
Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol
Description
Wild type E17.5 hearts
Data processing
Global and local normalization and data soothing in R (version, 2.10.1) was performed Peaks were detected using ChIPOlte and in-house algorithms, Correct P-value 0.001
