GSM2901375: Baseline1_ZT3; Mus musculus; ChIP-Seq
For collections, mice were deeply anesthetized with isoflurane, killed by cervical dislocation, the eyes removed to prevent any postmortem light effect, and the livers removed for processing. Liver tissue from each mouse was cut with a scalpel into small pieces (~ 0.5 mm3 size) and crosslinked with a freshly made formaldehyde solution (a protease inhibitor [PI] cocktail tablet (Complete Mini, EDTA-free, Roche, Indianapolis, IN, USA) dissolved in 1% formaldehyde in PBS)  at a ratio of 10 ml solution/1 g of tissue by rotation (Mini-Tube Rotator, Fisher Scientific) at room temp for 5 minutes. The reaction was quenched by adding 2.5 M glycine to a final concentration of 0.125 M and rotating at room temp for another 5 minutes.  The suspension was centrifuged for 5 minutes at 4°C in  Beckman Coulter GPKR centrifuge (84RCF) and the precipitant washed with 10 ml of cold PBS with PI, spun down, and the supernatant removed. The tissue was then homogenized on ice with a glass Dounce homogenizer (loose) for cell disruption in 5 ml PBS with PI, centrifuged 5 minutes at 1000 rpm at 4°C, re-suspended in 5 ml of Cell Lysis Buffer (5 mM PIPES, 85 mM KCL, 0.5% IGEPAL, 1 PI tablet) and incubated on ice for 15 minutes.  The tissue was then homogenized on ice with Dounce glass homogenizer (tight) with 10 strokes. Suspension was centrifuged for 5 minutes at 2000 g at 4°C, the supernatant removed, and isolated nuclei were checked under a microscope and then frozen at -80°C until sonicated.  Isolated nuclei were re-suspended in SDS Shearing Buffer (10 mM Tris HCl, 0.25% SDS, 1 mM EDTA, 1x Halt protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA), approximately 0.15 mg tissue per 1 µl of the buffer, and sonicated for 10 min (Duty Cycle 2%, Intensity 3, Peak Incident Power 105 Watts, Cycles per Burst 200).  Initial experiments were performed with isolated DNA to ensure a size range of approximately 200-700 bp.  After sonication, the sheared chromatin was centrifuged 5 minutes at 4°C at 10,000 RCF (Forma Scientific Micromax benchtop centrifuge) and supernatant was transferred to a new tube. Next, 100 µl of the chromatin prep (approximately 17 µg of DNA), was diluted with 200 µl of 1.5 x ChIP buffer (75 mM Tris HCl, 0.21 M NaCl, 1.5 mM EDTA, 1.5 % Triton-X, 0.15 % Deoxyholate) with PI and 3 µl of each sample was saved as input.  3 µl of histone H3 trimethyl Lys4 antibody (H3K4me3, Active Motif, Carlsbad, CA) was added to each experimental sample and the samples were incubated overnight at 4°C with rotation.  The chromatin prep from each liver was processed separately. Magnetic beads (Magna ChIP™ Protein A+G Magnetic Beads, Millipore), 20 µl per sample, were washed twice with 2 mL of 1x CHIP buffer (50 mM Tris HCl, 0.14 M NaCl, 1 mM EDTA, 1 % Triton-X, 0.1 % Deoxyholate) with PI and returned to an original volume of 20 µl.  The magnetic beads were added to each 100 µl sample and incubated for 2 hours at 4°C with rotation. The beads were then washed twice with 1 ml ChIP buffer (without PI) and incubated for 10 min at 4°C with rotation, followed by two washes with NaCl ChIP buffer (50 mM Tris HCl, 0.5 M NaCl, 1 mM EDTA, 1 % Triton-X, 0.1 % Deoxyholate), two washes with LiCl wash buffer (10 mM Tris HCl, 0.25 M LiCl, 1 mM EDTA, 0.5 % NP-40, 0.5 % Deoxyholate) and two washes with TE buffer (10 mM Tris HCl, 1 mM EDTA). After washing, 62.5 µl of TES buffer (50 mM Tris HCl, 1 % SDS , 10 mM EDTA) were added to each sample and the samples were incubated at 65°C for 10 minutes with mixing. The supernatants from each sample were saved in screw top cap vials.  The incubation step was then repeated and the supernatants combined. TES (113 µl ) was added to the input samples (total of 12 µl after combining samples from 4 livers). Next, 5.2 µl of 5 M NaCl and 6.25 µl of RNase A (4 µg/µl) were added to 125 µl of IP and input samples. The samples were then incubated for 30 - 60 min at 37°C.  After incubation, 5 µl of proteinasae K (20 µg/µl) was added to all samples and the samples decrosslinked overnight at 65°C.  After decrosslinking DNA from proteins, each DNA sample was purified with the MinElute PCR Purification Kit ( Qiagen, Valencia, CA ),  according to manufacturer's protocol and the samples representing each time point pooled. Illumina TrueSeq kit following manufacture's protocol
GEO Accession
GSM2901375