title: GSM1607378: H3K56ac ChIP-Seq Sirt6 Sirt6 KO RA Treated; Mus musculus; ChIP-Seq
library_construction_protocol: Chromatin immunoprecipitation (ChIP) steps were performed using a modified version of our previous protocols (e.g., Ram & Goren et. al. (2011) Combinatorial Patterning of Chromatin Regulators Uncovered by Genome-wide Location Analysis in Human Cells. Cell. 147 (7). and Garber & Yosef et. al. (2012) A High-Throughput Chromatin Immunoprecipitation Approach Reveals Principles of Dynamic Gene Regulation in Mammals. Molecular Cell 47 (5).) adapted to the Bravo liquid handling platform (Agilent). Cells were crosslinked using formaldehyde (1%, 37oC for 10 min), and then quenched with glycine (5min at 37oC). The fixed cells were lysed in Lysis buffer (1% SDS, 10mM EDTA and 50mM Tris-HCl pH 8.1, containing protease inhibitors (Roche, 04693159001). Chromatin was sheared using Covaris E-220 at 4oC to a size range between 200 and 800 bp. 1 to 5µg of antibody were incubated 2 hours with a mix of Protein-A and Protein-G Dynabeads (Invitrogen, 100-02D and100-07D, respectively) in blocking buffer (PBS supplemented with 0.5% TWEEN and 0.5% BSA). Beads were washed added to the chromatin lysate, and incubated overnight. Next, samples were washed 6 times with RIPA buffer, twice with RIPA buffer containing 500mM NaCl, twice with LiCl buffer (10mM TE, 250mM LiCl, 0.5% NP-40, 0.5% DOC), twice with TE (10Mm Tris-HCl pH 8.0, 1mM EDTA), and then eluted in ChIP-Elution Buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris Hcl pH 8.0) at 65oC, 4 hours, and then treated with RNaseA (Roche, 11119915001) for 30 min and Proteinase K (NEB, P8102S) for 2 hours. Illumina Library construction reactions were performed as we described previously (Garber & Yosef et. al. 2012). SPRI (Solid-phase reversible immobilization; AMPure XP beads (Agencourt)) cleanup steps were done using the Bravo liquid handling platform (Agilent) and a modified version of (Fisher et al., 2011). After the final ChIP step, 120µl SPRI was added to the reverse-crosslinked samples, pipette-mixed 15 times and incubated for 2 minutes. Supernatant was separated from the beads using a 96-well magnet for 4 minutes. Beads were washed on the magnet with 70% ethanol and then air dried for 4 minutes. DNA was eluted in 40µl EB buffer (10 mM Tris-HCl pH 8.0) by pipette mixing (25 times). The remainder of the library construction process (DNA end-repair, A-base addition, adaptor ligation and enrichment) the SPRI cleanup involves addition of PEG buffer (20% PEG and 2.5 M NaCl) to the reaction products. Next, plates were transferred to a magnet plate, incubated for 4 minutes and supernatant removed. Beads are washed on the magnet with 150µl 70% ethanol and air dried for 4 minutes. The DNA is eluted with 40µl of EB buffer (mixing 25 times). Reagent kits are prepared in advance for all enzymatic steps (New England Biolabs). The DNA end-repair was performed by adding 27µl of a master mix (17µl master mix (5µl T4 buffer, 5µl BSA-1mg/ml, 5µl ATP-10mM -2µl dNTPs 10 mM)) plus 5µl T4 PNK enzyme, 5µl T4 polymerase (3 units) to each well. Samples were incubated in a thermal cycler at 12oC for 15 min, 25 oC for 15 min, and finally cooled to 4 oC. The SPRI bead clean up method was used to purify the product (147µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 40µl EB). The A-base addition was performed by adding 20µl master mix (17µl A-base add mix, 3µl Klenow to each well and incubated at 37oC for 30 min in a thermal cycler. Product was purified using 132µl PEG buffer and eluted in 19µl EB. Adaptor ligation: was performed by adding 34µl of a master mix (29µl 2x DNA ligase buffer, 5µl DNA ligase) and 5µl indexed oligo adaptors (0.75 uM ) followed by 15min incubation in 25oC in a thermal cycler. Next, ligated products were purified using SPRI bead clean up with size selection (15.5µl of PEG buffer) and eluted in 40µl EB.  The final step of PCR enrichment was was performed by adding 10µl of a master mix (2µl Forward/Reverse Index Primer, 0.5µl dNTP mix, 5µl 10x PfuUltra Buffer, 1µl PfuUltra-II Fusion, 1.5µl Nuclease free water) to each well. Using a thermal cycler, programed to 95C for 2 min, followed by 16 cycles of (95oC for 30 sec, 55oC for 30 sec, 72oC for 60 sec), and a final incubation at 72oC for 10 min. The last SPRI clean up was coupled to size selection (35µl SPRI beads were added to each sample and eluted in 40µl).
attributes.tag: GEO Accession
attributes.value: GSM1607378