GSM2053718: BLRP Control ChIP-Seq 1; Mus musculus; ChIP-Seq
Mice were first euthanized with Isoflourane, livers liver was then macerated in PBS containing 1% formaldehyde and protease inhibitor cocktail 1 (PIC1, comprised of 1μg/ml leupeptin, 1.4μg/ml pepstatin, 0.2mg/ml PMSF (Sigma), 1mM EGTA, 1mM EDTA) for 10 minutes with gentle rotation at room temperature. Crosslinking was then stopped by the addition of glycine (0.125M) and samples were further incubated for 5 minutes at room temperature. Fixed livers were then placed on ice and then centrifuged (2,000 x g for 10 minutes) and the pellet resuspended in 8ml of PBS containing protease inhibitor cocktail. Resuspended pellets were gently homogenized and again centrifuged as above. The resulting pellet was resuspended in 5ml lysis buffer (5mM PIPES, pH 8.0, 85mM KCl, 0.5% NP-40 alternate) supplemented with protease inhibitor complex 2 (PIC2, 10μg/ml leupeptin, 5μg/ml pepstatin, 0.2mg/ml PMSF, 50μg/ml ALLN, Sigma). Nuclei were released after 30 strokes using a Dounce homogenizer and collected after centrifugation as above. Pellets were resuspended in 6ml homogenization buffer (10mM HEPES, pH 7.6, 25mM KCl, 1mM EDTA, 1mM EGTA, 1M Sucrose, 10% glycerol, 0.15mM spermine, supplemented with PIC1) and layered onto 3ml of the same buffer. Nuclei were then pelleted at 26,000rpm for 1 hour (Beckman SW41 rotor) and stored at -80ºC. Nuclear pellets were re-suspended in 0.3ml nuclear lysis buffer (50mM Tris pH 7.6, 10mM EDTA and 1% SDS), and diluted with 0.6ml immunoprecipitation (IP) dilution buffer (0.01% SDS, 1.1% Triton x100, 167mM NaCl, 16.7mM Tris pH 7.6, 1.2mM EDTA). For sonication, 0.3ml (1/3) of nuclear lysate was sonicated for 25-30 cycles 30 seconds on 30 seconds off at 4ºC with BioRuptor twin sonicator (Diagenode). Sonicated chromatin was then further diluted to 1ml with IP dilution buffer, which is sufficient for three ChIP reactions. The BLRP antibody (Genescript) was conjugated to Protein G Dynabeads (Life Technologies) in PBS containing 0.5% BSA overnight at 4ºC with gentle rotation. Sonicated DNA (0.33ml made up to 1ml in IP dilution buffer) was then incubated overnight with Protein G-BLRP beads at 4ºC with gentle rotation. For input samples, 10% was used. Samples were then washed twice with wash buffer I (20mM Tris, pH 7.4, 150mM NaCl, 0.1% SDS, 1% Triton X100, 2mM EDTA), three times with alternate wash buffer III (10mM Tris, pH 7.4, 250mM LiCl, 1% NP-40 alternate, 0.7% Deoxycholate, 1mM EDTA), two washes with 0.2% Triton X100 TE buffer and two final washes with 50mM NaCl containing TE buffer. Chromatin was recovered and DNA from IP and input was isolated after reverse crosslinking (incubating at 65ºC overnight in 0.3M NaCl and Proteinase K digestion) and DNA purified using gel/PCR extraction kit (Clontech). ChIP-Seq: Sequencing libraries were prepared from collected DNA by blunting, A-tailing, adapter ligation using the Kapa Library Prep kit according to manufacturers instructions.
GEO Accession
GSM2053718