GSM1647868: mm_input_DNA; Mus musculus; ChIP-Seq
ChIP-seq library:      Frontal cortices from 4-5 male mice (F1 Hybrids of of C57BL6N/129SvEv) at 6-week old were used for a single ChIP experiment. After micro-dissected, the frontal cortex was minced into small pieces (~1 mm3) and immediately cross-linked in 1% formaldehyde for 20 minutes at room temperature. 125 mM glycine was added to quench crosslinking for 5 min at room temperature. The tissue was washed twice in cold phosphate-buffered saline (PBS) containing halt protease inhibitor cocktail (Pierce Biotechnology, Inc.), centrifuged at 850g for 10 min at 4 °C, the tissue pellets were either stored at -80°C until use or processed immediately. Tissue pellets were homogenized (Fisher Scientific™ PowerGen™ Model 125 Homogenizer) in 1 ml ice-cold cell lysis buffer I (50 mM HEPES-KOH, 1 mM EDTA, pH 8.0, 140 mM NaCl, 0.25% Triton X-100, 0.5% NP-40 and 10% glycerol and halt protease inhibitor cocktail).  The resulting material was rocked at 4 °C for 10 min, filtered through a 70um/100um cell strainer and pelleted by centrifugation at 850g for 10 min at 4 °C. The pellet was re-suspended in 1ml ice-cold cell lysis buffer II (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 200 mM NaCl and 0.5 mM EGTA, pH 8.0 and halt protease inhibitor cocktail) and rocked at 4°C for 10 min, then re-pelleted. The pellet was re-suspended in 130ul sonication buffer (50mM Tris-HCl, pH 8.0, 10mM EDTA, 0.5% SDS and halt protease inhibitor cocktail), rocked at 4°C for 10 min and then sonicated (Covaris) into 200–700pb. After centrifugation at 13000 r.p.m. for 30 min at 4 °C, the supernatant was transferred to a new tube and diluted in 4 volume modified RIPA buffer (175mM NaCl, 1.25% triton X-100, 0.125% Na-DOC) with halt protease inhibitor cocktail. Then 10% of the supernatant was stored as input material. The rest was incubated overnight at 4 °C with 30 μl of Dynal protein G magnetic beads (Life technologies) that had been pre-incubated with 4 μg of Anti-Egr1. Beads were washed five times with 180 μl of ice-cold RIPA buffer (10 mM Tris-HCl, pH 8.0 , 1 mM EDTA, pH 8.0, 140 mM NaCl, 1%  Triton X-100, 0.1% SDS and 0.1% Na-DOC), twice with 180 μl of RIPA-500 buffer (10 mM Tris-HCl, pH 8.0 , 1 mM EDTA, pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% SDS and 0.1% Na-DOC), twice with 180 μl of ice-cold LiCl wash buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 250 mM LiCl, 0.5% NP-40 and 0.5% Na-DOC), and once with 180 μl of ice-cold 1× TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Then the 200ul elution buffer (10 mM Tris-HCl, pH 8.0, 5 mM EDTA, pH 8.0, 300 mM NaCl and 0.5% SDS) with 4 ul RNase Cocktail (AM2286) was added to the immunoprecipated material. The saved input material was adjusted to 200ul by adding elution buffer with 4 ul RNase Cocktail. These samples were incubated at 37 °C 30min, followed by adding  4 ul proteinase K (AM2546), treated at 65°C overnight. The genomic DNA fragments were recovered by AMPure XP beads (2.3x ratio, Beckman Coulter, Inc.), and the library construction was performed as described previously. DNA in the range 270 - 600 bp was recovered by Pippin Prep(Sage Science), after size distribution assessment by Agilent bioanalzer and quantification by qPCR (Kapa Library quantification kit),  libraries were subjected to 150-bp paired-end read sequencing on the Illumina HiSeq 2000 platform. To extract genomic DNA from paraffin block for human cortex gray and white matter, xylene was first added to these blocks, vortexed for 1 minutes and gentle shaked at RT for 5-15min. The supernatant was discarded after centrifugation, the pellets were washed with absolute ethanol twice to remove xylene, dried them on bench. 1ml lysis solution was added  (50 mM Tris, 25 mM EDTA, 100 mM NaCl, 0.5% Tween-20/SDS, pH 8), inverted every 30 minutes until the pellets were completely lysed,  40 μl 20mg/ml proteinase K was added, Incubated overnight at 55 ºC.  After 1-hour 90 ºC incubation, 5ul RNase cocktail was added and incubated at 37 ºC for 30minutes. Phenol-cholorophorm method was used to extract the DNA from samples