title: GSM8126209: H3K27Ac Hypoxia; Homo sapiens; ChIP-Seq
library_construction_protocol: Approximately 1 × 10^7 cells were harvested by scraping the plates and transferring them into a 15 ml tube. Cells were centrifuged at 2,000 g at 4°C for 5 minutes. The supernatant was discarded, and cells were lysed in 10 ml ice-cold cell lysis buffer (20 mM Tris-HCl pH 8.0, 85 mM KCl and 0.5% NP-40) containing 1× proteinase inhibitor (PI). The lysed cell suspension was rotated at 8 r.p.m. at 4°C for 15 minutes using a hula mixer. Nuclei were counted, and approximately 3 × 10^7 nuclei were pelleted by centrifugation at 2,000 g at 4°C for 5 minutes. The pelleted nuclei were resuspended in 2 ml nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, and 1% SDS) containing 1× PI. Sonication was performed in a 15 ml tube with sonication beads with Bioruptor pico, alternating 30 seconds "on" and 30 seconds "off" for a total of 30 cycles at 4°C. The quality and size range of the sheared DNA were assessed using gel electrophoresis to confirm the desired fragmentation.For chromatin fraction quality assessment, 10 µl of the fraction was treated with proteinase K and purified using a PCR clean-up kit, followed by gel electrophoresis. 40 µl of protein A dynabeads were blocked in 500 µl of RIPA-150 (50 mM Tris-HCl pH 8.0, 0.15 M NaCl, 1 mM EDTA pH 8.0, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate) containing 3% BSA at 8 r.p.m. at 4°C for 1 hour in a 1.5 ml tube using a hula mixer. 2 µg of antibody (H3K4me1, H3K4me3, H3K27Ac) was pre-bound to the protein A dynabeads in 500 µl of RIPA-150 at 8 r.p.m. at 4°C for 2 hours in a 1.5 ml tube using a hula mixer. 700 µl of chromatin fraction was diluted with 6.4 ml of ChIP dilution buffer (50 mM Tris-HCl pH 8.0, 0.167 M NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate) and 2 ml of RIPA-150. The pre-bound dynabeads were mixed with the diluted chromatin fraction. The mixture was then incubated at 8 r.p.m. at 4°C overnight in a 15 ml tube using a hula mixer. The beads were washed sequentially with 500 µl of RIPA-150, RIPA-500 (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 1 mM EDTA pH 8.0, 0.1% SDS, 1% Triton X-100 and 0.1% sodium deoxycholate), RIPA-LiCl (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 1 mM EDTA pH 8.0, 0.1% SDS, 1% Triton X-100 and 0.1% sodium deoxycholate) and TE buffer. Between each wash, the beads were transferred to a new tube to minimize non-specific binding. DNA was eluted in elution buffer containing RNase A. The eluted sample was transferred to a new tube and digested with proteinase K at 56°C overnight. Purified DNA followed by DNA concentrator-5 (Zymo). Library construction was carried out using KAPA HyperPrep Kits.