GSM5885578: 3-vegf-0h-input; Homo sapiens; ChIP-Seq
Chromatin immunoprecipitation (ChIP) was performed as previously described (PMID: 32125007). In brief, 10 million HUVECs were cross-linked with 1% formaldehyde for 10 min and then neutralized with 0.2 M glycine for 5 min. Collected cell pellets were re-suspended in SDS lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 1% SDS, 1 mM EDTA) with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich), and fragmented by a Picoruptor (40 cycles, 30 sec on/30 sec off; Diagenode, Liege Science Park, Belgium). The sonicated solution was diluted with ChIP dilution buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) to achieve 1 to 5 dilution. The specific antibody was bound to magnetic Dynabeads M-280 (Thermo Fisher Scientific) and applied to the diluted, sonicated solution for immunoprecipitation. Antibody against H3K36me3 (MAB Institute, Inc. Hokkaido, Japan) was used. DNA sonicated to an average size of 0.5 kb was used for the ChIP-seq library preparation. ChIP samples were processed for library preparation with a KAPA Hyper Prep Kit (Kapa Biosystems Inc., Wilmington, MA), according to the manufacturer's instructions. Deep sequencing was performed on a HiSeq 2500 sequencer (Illumina Inc., San Diego, CA) as single-end 50 base reads. Libraries were prepared according to Illumina's instructions.