title: GSM1611583: Modified ChIP against H3K27ac; Mus musculus; ChIP-Seq
library_construction_protocol: For each ChIP 100 million cells were used. As a control, CGR8 Embryonic Stem Cells expressing no V5 tag were used. Cells were cross-linked and washed twice with cold 1x PBS and collected by centrifugation. Cells were resuspended  in LB1 (50mM Hepes-KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% Triton-X-100 . After 10 minutes incubation, cells were pelleted by centrifugation and resuspended in LB2 (10 mM Tris-HCl, pH8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA. After 10 minutes incubation, cells were pelleted and resuspended in freshly prepared LB3 (10mM Tris-HCl pH8.0, 100mM NaCl, 1mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine and sonicated on a Soniprep 150(MSE). For quality control of the sonication, it was verified whether the DNA fragment size was between 100 and 1000bp. V5-Agarose beads were blocked 1 hr with 0.5 mg/ml BSA. The chromatin was incubated overnight with the blocked V5-Agarose beads. The following day the beads were washed 5 times with RIPA buffer and eluted with elution buffer 50mM Tris-HCl pH8.0 10mM EDTA and 1% SDS for 30 minutes. Samples were de-cross-linked overnight at 65 degrees celcius and treated the following day with proteinase K for one hour at 45 degrees celcius. DNA was extracted by phenol/chloroform/isoamyl extraction and precipitated with ethanol and resuspended in water. The library was prepared according to the illumina protocol. Briefly 10 ng of DNA ChIP material was end-repaired, ligated to adapters, size selected on gel (200 bp +-25 bp range) and PCR amplified using Phusion polymerase with 30 seconds incubation at 98 ᴼC, 18 cycles of 10 second 98 ᴼC , 30 seconds 65 ᴼC, 30 seconds 72ᴼC. Followed with 5 minutes of final extension at 72 ᴼC.  Cluster generation was performed using the illumina genome analyzer II platform.
attributes.tag: GEO Accession
attributes.value: GSM1611583