GSM2586793: exp4_CREB1_vehicle; Mus musculus; ChIP-Seq
Shearing of chromatin was carried out by the manufacturer's protocol of truChIP kit (PN 520075, Covaris, Woburn, Massachusetts). All Covaris buffers were provided in kit. Confluent monolayers mpkCCD cells cultured on 100 mm permeable supports of transwell system were treated with dDAVP (0.1 nM) or vehicle for 30 minutes and then washed in ice-cold PBS followed by cross-linking in 1.1% formaldehyde in Covaris Fixing Buffer (5.5 ml) for 5 minutes at room temperature. Cross-linking was terminated by the addition of 0.3 ml of 1X Covaris Quenching Buffer with rocking. The buffer solution was aspirated and ice-cold PBS was added to each plate. Cells were scraped, spun down (200xg, 5 minutes), and washed twice with ice-cold PBS. Cell pellets were resuspended in 1 ml of 1X Covaris Lysis Buffer for 10 minutes at 4°C with rocking.  Nuclei were pelleted by spinning at 1,700xg for 5 minutes at 4°C. Nuclear pellets was resuspended in 1 ml of 1X Covaris Wash Buffer and incubated for 10 minutes at 4°C with rocking. Samples were spun at 1,700xg for 5 minutes at 4°C. Nuclear pellets were washed twice with 1 ml of Covaris Non-ionic Shearing Buffer and spun at 1,700xg for 5 minutes at 4°C. Nuclei were resuspended in 1 ml of Covaris Non-ionic Shearing Buffer. Two 20 ul aliquots of the resuspended pellets were removed prior to shearing to be used as subsequent unsheared controls for immunoblotting and agarose gel electrophoresis. The remaining samples were transferred to AFA tubes (TC12 x 12 mm). Samples were sheared using a Covaris S2 SonoLAB Single system for 6 minutes (Duty cycle: 5%; Intensity: 4; cycle: 200). Total nuclear protein concentration of each sample in mg/ml was determined with the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL).  To test shearing efficiency, a 20 ul aliquot from each sample was obtained after shearing. The aliquots from the unsheared sample and the sheared sample, were incubated with 100 ul 1X TE buffer, 10 ul 10% SDS, and RNAse A (10 ug/ul, Cell Signaling Technology, Danvers, Massachusetts) for 30 minutes at 37°C. After adding proteinase K (2 ug), the samples were incubated overnight at 65°C. The DNA was purified using DNA Clean & Concentrator columns (Catalog #: D4013, Zymo Research, Irvine, CA) and ran on an E-gel EX 2% agarose (Invitrogen, Carlsbad, CA). Target fragment sizes for chromatin immunoprecipitation were in the range of 150-300 bp.  Immunoprecipitaiton was performed by the manufacturer's instruction (SimpleChIP protocol #9003, Cell Signaling Technology). 'Input' sample (10 ul) was obtained prior to immunoprecipitation.  Chromatin Immunoprecipitation was performed using 700-800 ug of sheared chromatin in 800 uL of 1X Covaris Non-ionic Shearing Buffer with 4 μg of anti-CREB1, anti-CEBPB or anti-rabbit IgG antibodies (as negative control). The mixtures were incubated overnight at 4°C with gentle rotation, followed by incubation with ChIP Grade Protein G Magnet Beads (Catalog #: 9006, Cell Signaling Technology) for 2 hours at 4°C with rotation. The beads were washed a total of four times, including three low salt washes followed by one high salt wash for 5 minutes each at 4°C. Immunoprecipitated chromatin was eluted from the beads with 150 ul of 1X ChIP Elution Buffer for 30 minutes at 65°C in a thermomixer. To reverse crosslinks, 6 ul of 5 M NaCl and 2 ul of 20 ug/ul  Proteinase K were added to the chromatin followed by incubation at 65°C overnight. DNA was purified according to the manufacturer's instruction. Sequencing libraries were prepared according to the manufacturer's instruction (NuGEN Ovation SP Ultralow library system kit). Adaptor-conjugated iibraries were amplified by PCR. The amplified libararies were measured.  A fixed amount of the total measured DNA was used for sequencing in an Illumina Hiseq2000 instrument to obtain single read data.  Libraries were sequenced using an Illumina HiSeq2000, obtaining 1- by 50-bp reads.
GEO Accession
GSM2586793