title: GSM8238386: H3K27ac_mESCs_KO_rep1; Mus musculus; ChIP-Seq
library_name: GSM8238386
library_construction_protocol: mESCs were cross-linked by incubating them initially in Gold (Diagenode), and then they were further crosslinked in DPBS (Gibco, Cat #14190094) supplemented with 1% formaldehyde (Sigma-Aldrich; Cat #252549) for 10 min rocking at room temperature. Cross-linking was stopped using 125 mM Glycine (Sigma-Aldrich, Cat #G8790) rocking for 5 minutes at room temperature. Cell nuclei were isolated by incubating cell pellets in a buffer containing 5mM Pipes pH 8, 85mM KCl and 0.5% NP40, and later incubated on ice for 10 minutes followed by centrifuge at 4000 rpm for 5 minutes at 4ºC. Nucleus were lysed in a buffer containing 1% SDS, 10mM EDTA and 50 mM Tris-HCl pH 8.1 and incubated on ice for 10 min. Samples were sonicated 25 cycles (30´´on/30´´off) and SDS was removed by incubating the sonicated sample on ice for 1 hour and later centrifuged for 15 at 4ºC and 16000g. 60μg of chromatin was incubated with 10 μg of ANTI-FLAG® M2 (Sigma-Aldrich, Cat #F1804). Samples were pulled down by a 50/50 mix of Dynabeads A (Invitrogen, Cat #10002D) and G (Invitrogen, Cat #10003D). Samples were washed 4 times in 0.1% SDS, 1% Triton-X, 2mM EDTA, 20mM Tris-HCl pH 8 and 150mM NaCl; followed by 1 wash in TE pH 8. For H3K4me1 and H3K27Ac ChIP-seqs, cells were crosslinked only with PBS and 1% formaldehyde as described, and cross-linking solution was reverted by Glycine as previously described. Cells were lysed by resuspending cell pellet in ice-cold ChIP buffer (1 volume of SDS buffer [100mM NaCl, 50mM Tris-HCl pH 8.1, 5mM EDTA pH 8, 0.5% SDS] and 0.5 volume of triton dilution buffer [100mM Tris-HCl pH 8.6, 100mM NaCl, 5mM EDTA pH 8, 5% Triton X-100]) supplemented with protease/phosphatase inhibitor cocktail (1X cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche, Cat #11873580001), 1 mM PMSF, 1mM Sodium Ortovanadate and 20 mM β-Glycerol phosphate), and samples were sonicated using sonication beads (Diagenode; Cat #C01020031) and the Bioruptor Pico Sonicator (Diagenode; Cat #B01060010) for 20 cycles (each cycle 30 seconds on/30 seconds off) or till DNA fragments have 100-300bp size. Chromatin was quantified, and 30 μg of chromatin was incubated with every 5μg of antibody rotating for 16 hours (or overnight) at 4ºC. Samples were pulled down by incubation with Protein A-Sepharose™ CL-4B (previously hydrated and blocked with 0.05% BSA) for 3h at 4ºC in rotation. Sample was washed with a low salt washing buffer (50mM HEPES pH 7.5, 140mM NaCl and 1% Triton) three times, one wash with a high salt wash buffer (50mM HEPES pH 7.5, 500mM NaCl and 1% Triton) and one wash with TE pH 8 (10mM Tris-HCl pH8, 1mM EDTA). Samples were then eluted in 1% SDS and 100mM NaHCO3. Samples were descrosslinked by incubating for 16 hours (or overnight) at 65ºC shaking at 450 rpm, and were treated with Proteinase K for 1h at 45ºC. DNA was purified using QIAquick PCR Purification Kit (Qiagen, Cat #28106), following the manufacturer's instructions. Libraries prepared according to manufacturer's instructions (SE 50bp reads)