title: GSM4818175: H3K27ac-NHP-LvE-4-17870; Macaca mulatta; ChIP-Seq
library_construction_protocol: Cell pellets were dissolved in PBS and crosslinked in solution by using formaldehyde (1% final volume, shaking 10 min at room temperature). Glycine (0.125 M) was added to quench the reaction. Cells were then washed two times with cold PBS. Cell pellets were lysed in a volume of 110µl using lysis buffer (20 mM HEPES pH 7.6, 1% SDS, 1×Protease Inhibitor Cocktails). Samples were sonicated using the Biorupter Pico (Diagenode) with eight cycles (30 s on/30 s off). Afterward, the samples were spun at 16,000×g for 5 min at room temperature and the supernatant was stored at -80°C. Chromatin from 100,000 cells was used for ChIPmentation. ChIPmentation was performed as described by Schmidl et al (*), with several modifications. In short, The chromatin was incubated overnight at 4°C rotating in dilution buffer (1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8, 167 mM NaCl), 1×Protease Inhibitor Cocktail (Roche) and 1µg of antibody [H3K27ac (C15410196, Diagenode)] in a total volume of 300 µl. The next day per ChIP 10µl protein A Dynabeads and 10µl protein G Dynabeads  (both Invitrogen) were added. Beads were washed before use with dilution buffer (+0.15% SDS,+0.1% BSA) and incubated with the chromatin and antibody mix for 60 min at 4°C rotating. Afterward, the beads were washed at 4°C once with ChIP wash buffer 1 (2 mM EDTA, 20 mM Tris pH 8, 1%Triton X-100, 0.1% SDS, 150 mM NaCl), twice with ChIP washbuffer 2 (2 mM EDTA, 20 mM Tris pH 8, 1% Triton X-100, 0.1%SDS, 500 mM NaCl), and twice with TE (1 mM EDTA, 10 mM TrispH 8). Beads were resuspended in 24 µl Tagment DNA buffer ( Nextera) and 1µl Tn5 enzyme (produced in-house) and incubated for 10 min at 37°C with 550 rpm shaking. Afterward, the beads were washed twice with WBI (20 mM HEPES, 150 mM NaCl, 0.1% SDS, 0.1% DOC,1% Triton X-100, 1 mM EDTA, 0.5 mM EGTA) and twice with WBIV (20 mM HEPES, 1 mM EDTA, 0.5 mM EGTA) with 5 min rotating at room temperature in between. Samples were decrosslinked for 1 h at 55°C 1,000 rpm shaking followed by an overnight incubation at 65°C using elution buffer (0.5% SDS, 300 mM NaCl,5 mM EDTA, 10 mM Tris pH 8) and proteinase K. Samples were incubated one additional hour the next day with elution buffer and proteinase K for 1 h at 55°C. The samples were purified using 2×SPRI AMPureXP beads. qPCR was used to determine the sufficient amount of PCR cycles needed to amplify the library. The libraries were amplified using the KAPA HiFi Hotstart Ready Mix (KAPA Biosystems) and Nextera Index Kit 1 (i7) and 2 (i5) primers (Illumina). Amplified libraries were purified using a 0.65× SPRI AMPureXP beads incubation followed by a 1.8× SPRI AMPureXP beads incubation. Library concentration was measured using the KAPA Library Quantification Kit (KAPA Biosystems); library size  was determined using the BioAnalyzer High Sensitivity DNA Kit (Agilent). Sequencing was performed using an Illumina HiSeq 2000, and 50-bp paired-end reads were generated. ChIP-seq libraries were prepared from precipitated DNA of  1 million cells for the histone tail modifications. End repair was performed using Klenow and T4 PNK. A 3' protruding A base was generated using Taq polymerase and adaptors were ligated. The DNA was loaded on E-gel and a band corresponding to ~300 bp (ChIP fragment + adaptors) was excised. The DNA was isolated, amplified by PCR and used for cluster generation and sequencing on the HiSeq 2000 or NextSeq (Illumina). RNA-seq: Total RNA was isolated with the RNeasy RNA extraction kit with on-column DNaseI treatment (Qiagen), and the concentration was measured with a Qubit fluorometer (Invitrogen). Ribosomal RNA was removed by Ribo-Zero rRNA Removal Kit (Epicentre) according to manufacturer instructions. 16 µl of purified RNA was fragmented by addition of 4 µl 5× fragmentation buffer (200 mM Tris-acetate pH 8.2, 500 mM potassium acetate and 150 mM magnesium acetate) and incubated at 94℃ for exactly 90 s. After ethanol precipitation, fragmented RNA was mixed with 5 µg random hexamers, followed by incubation at 70℃ for 10 min and chilling on ice. We synthesized the first-strand cDNA with this RNA primer mix by adding 4 µl 5× first-strand buffer, 2 µl 100 mM DTT, 1 µl 10 mM dNTPs, 132 ng of actinomycin D, 200 U SuperScript III, followed by 2 h incubation at 48℃. First strand cDNA was purified by Qiagen mini elute column to remove dNTPs and eluted in 34 µl elution buffer. Second-strand cDNA was synthesized by adding 91.8 µl, 5 µg random hexamers, 4 µl of 5× first-strand buffer, 2 µl of 100 mM DTT, 4 µl of 10 mM dNTPs with dTTP replaced by dUTP, 30 µl of 5× second-strand buffer, 40 U of Escherichia coli DNA polymer¬ase, 10 U of E. coli DNA ligase and 2 U of E. coli RNase H, and incubated at 16℃ for 2 h followed by incubation with 10 U T4 polymerase at 16℃ for 10 minutes. Double-stranded cDNA was purified by Qiagen mini elute column and used for library preparation as described in the KAPA HyperPrep protocol.