title: GSM5097090: H3K27ac_HC0_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: Freshly dissected tissue was chopped by a razor blade and rapidly incubated in 1.5 mL PBS containing 1% formaldehyde for 10 minutes at RT. To stop fixation, glycine was added (0.125 M final concentration). Dorsal hippocampi from 4 mice were pooled per sample. Tissue samples were then processed as described in Chatterjee et al. 2018, and sonicated using the Diagenode Bioruptor (30 s ON-30 s OFF at High Power x 35 cycles). Sonicated chromatin was centrifuged 10 min at 14000xg, the supernatant collected and diluted 1:10 in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl, pH 8.1, 167 mM NaCl). A fraction of the supernatant (50 mL – 10%) from each sample was saved before immune-precipitation for 'total input chromatin'. Supernatants were incubated overnight (4°C) with 1/1000 primary antibody H3K9/14ac (Diagenode C15410200) and H3K27ac (ab4729), followed by protein A Dynabeads (Invitrogen) for 2 hours at RT. After several washes (low salt, high salt, LiCl and TE buffers), the resulting DNA-protein complexes were eluted in 300 mL elution buffer (1% SDS, 0.1 M NaHCO3). The crosslinking was reversed (overnight at 65°C) and the DNA was purified with RNAse (30 min, 37°C), proteinase K (2 hours at 45°C). DNA from the immunoprecipitated and input samples was isolated using Diagenode MicroChIP DiaPure columns with 20 mL nuclease-free milliQ water in low binding tubes. ChIP samples were purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified with the Qubit (Invitrogen). ChIP-seq libraries were prepared from 10ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (C05010014, Diagenode s.a., Seraing, Belgium), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5 prime ends were ligated to the 5 prime end of the genomic DNA, leaving a nick at the 3 prime end. The adaptors cannot ligate to each other and do not have single-strand tails, avoiding non-specific background. In the final step, the 3 prime ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (4 + 7 cycles). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter) to remove unincorporated primers and other reagents.