title: GSM1467635: WT74 H3K4me3; Mus musculus; ChIP-Seq
library_construction_protocol: Chromatin Preparation: Testes were initially isolated and stored at -80C. Tissue was tehn chopped and placed in a 15ml conical with DMEM. 37% formaldehyde was added toa final concentration of 1% and rocked for 12 minutes. 1.25 ml of glycine was added, rocked for 2 minutes then centrifuged. Tissue was then washed three times with 10ml sterile PBS. 1 ml cell lysis buffer(5 mM PIPES pH8, 85 mM KCl. Add igepal fresh each time to give a final concentration of 1% (10 μl/ml) plus protease inhibitors (Roche – 11 836 170 001)) was added and incubated for 15 min. Cells were then dounced 23 times, transferred to 1.5 ml tube and spun for 5 min at 2500G. Cells were then re-suspended in 200ul nuclear lysis buffer (50 mM Tris-HCl pH8, 10 mM EDTA, and 1% (w/v) SDS plus protease inhibitor) and incubated on ice for 30 minutes. Pellet was flash frozen in LN2, thawed slightly and sonicated (15 minutes, 30 seconds on, 1 minute off). PCR was used to check fragment size.  Chromatin was diluted with IP dilution buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1% (v/v) igepal, 0.25% (w/v) deoxycholic acid, 1 mM EDTA pH8 plus protease inhibitors). Aliquots of each sample were taken and incubated with one of the following antibodies overnight at 4 degrees C: 3ul of anti trimethyl histone H3 Lys 4 (H3K4me3, Millipore # 04-745). 15ul of each sample was also saved for input. Samples were incubated with antibody overnight at 4 degrees C. 15 ul magnetic Protein G beads (Cell Signaling 9006S) were added to each sample and incubated for 2 hours at 4. Beads were magnetized and washed 2x in IP dilution buffer, 1x in IP wash buffer 2 (100 mM Tris-HCl pH 9, 500 mM LiCl, 1% (v/v) igepal, 1% (w/v) deoxycholic acid), 2x in IP wash buffer 3 (100 mM Tris-HCl pH 9, 500 mM LiCl, 150 mM NaCl, 1% (v/v) igepal, 1% (w/v) deoxycholic acid).  Chromatin was eluted with 100ul elution buffer (50 mM NaHCO3, 1% (w/v) SDS) and vortexed at RT for 50 min. Beads were magnetized, supernatant (including input) was incubated at 67 degrees C O/N to reverse crosslinks. Qiagen's QiaQuick PCR purification Kit (Qiagen 28104) was used to purify DNA, and RT-PCR and qPCR was used to determine specificty of chromatin. Libraries were prepared according to directions stated O'geen et al. 2011 (PMID: 21913086)