title: GSM1692808: HE_Input; Mus musculus; ChIP-Seq
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
attributes.tag: GEO Accession
attributes.value: GSM1692808