ES derived Hemogenic endothelium (Bry.GFP+, Tie2+/Ckit+/CD41-)
chip antibody
H3K27ac Abcam ab4729
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For histone modification ChIP, nuclei of approximately 2x 10^6 sorted and crosslinked cells were isolated in hypotonic buffer A (10 mM Hepes, pH 8.0, 10 mM EDTA, 0.5 mM EGTA, 0.25 % TritonX100) and washed in buffer B (10 mM Hepes, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.25 % TritonX100). Chromatin was then sonicated in immunoprecipitation buffer I (25 mM Tris 1 M, pH 8.0, 150 mM NaCl, 2 mM EDTA, pH 8.0, 1 % TritonX100 and 0.25 % SDS) using a Bioruptor water bath (Diagenode). After centrifugation the sheared 200 – 500 bp chromatin fragments were diluted with 2x volume of immunoprecipitation buffer II (25 mM Tris, pH 8.0, 150mM NaCl, 2 mM EDTA, pH 8.0, 1 % TritonX100, 7.5 % glycerol), 5 – 10% of input chromatin was saved and the precipitation was carried out with remaining chromatin for 2 - 4 hours at 4 °C using 0.2 - 2 ?g of specific antibody (H3K9ac: Abcam ab4441 and Millipore ABE18, H3K27ac: Abcam ab4729, H3K4me3: Millipore 04-745, H3K27me3: Abcam ab6002) coupled to 15 ?l protein G Dynabeads (Dynal). Beads were washed with low salt buffer (20 mM Tris 1 M, pH 8.0, 150 mM NaCl, 2 mM EDTA, pH 8.0, 1 % TritonX100, 0.1 % SDS), high salt buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 2 mM EDTA, pH 8.0, 1 % TritonX100, 0.1 % SDS), LiCl buffer (10 mM Tris, pH 8.0, 250 mM lithium chloride, 1 mM EDTA, pH 8.0, 0.5 % NP40, 0.5 % sodium-deoxycholate) and TE pH 8.0 containing 50 mM sodium chloride. The immune complexes were eluted in 100 ?l elution buffer (100 mM NaHCO3, 1 % SDS) and, after adding 5 ?l of 5M sodium chloride and proteinase K, the crosslinks were reversed at 65°C overnight. DNA was extracted by using the Ampure PCR purification kit and ChIP quality was validated by realtime PCR. The libraries were prepared according to Illumina TruSeq DNA Sample Prep Kit