Cultured cells were fixed by adding 1% Formaldehyde (16% Formaldehyde (w/v), Methanol-free, Thermo Scientific) directly to the medium and incubating for 8 minutes. Glycine (150 µM) was added for 5 min to quench the formaldehyde. Cells were washed twice with ice-cold PBS 0.5% Triton-X100, scraped and collected by centrifugation (1000 × G for 5 min at 4 °C). The pellet was resuspended in 1400 µL of RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA pH 8, 1% Triton-X100, 0.5% Sodium deoxycholate, 1% SDS, 1% protease inhibitors) and transferred in a new eppendorf tube. The lysate was homogenized by passing through an insulin syringe, and incubated on ice for 10 min. The chromatin was sonicated for 3 min by using a 250 Digital Sonifier (Branson) with 0.7 s 'On' and 1.3 s 'Off' pulses at 40% power amplitude, yielding a DNA fragment size of 100 to 500 bp. The sample was kept ice-cold at all times during the sonication. The samples were centrifuged (10 min at 16000 × G at 4 °C) and supernatants transferred to a new eppendorf tube. 50 µL of sheared chromatin was set apart as “input”, and 5 µg of either rabbit anti-FLAG (Pierce), rabbit anti-Prox1 (ProteinTech), rabbit anti-p300 antibody (Santa Cruz) or rabbit anti-H3K9ac (Cell Signaling) antibody was added to the remainder of the chromatin. Samples were incubated overnight at 4 °C on a rotator. 20 µL Pierce Protein A/G Magnetic Beads (Life Technologies) were added to the samples and incubated at 4 °C for at least 5 hours. A/G Magnetic Beads were collected and washed 5 times with washing buffer (50 mM Tris-HCl, 200 mM LiCl, 2 mM EDTA, pH 8, 1% Triton, 0.5% Sodium deoxycholate, 0.1% SDS, 1% protease inhibitors), and twice with TE buffer. The A/G magnetic beads were resuspended in 50 µL of TE buffer, and 1.5 µL of RNase A (200 units, NEB, Ipswich, MA, USA) were added to the A/G beads samples and to the input, incubated for 10 minutes at 37 °C. After addition of 1.5 µL of proteinase K (NEB, 200 units) and overnight incubation at 65°C, the DNA was purified using 1.8 × volume of Agencourt AMPure XP (Beckman Coulter) according to the manufacturer’s specifications, and then eluted in 30 µL of TE buffer. Input and immunoprecipitated DNA was transformed into libraries using the NEBNext Ultra DNA library prep kit for Illumina (NEB), and amplified using KAPA HiFi HotStart ReadyMix for 14 cycles.
GEO Accession
GSM1831055