title: GSM4873123: 3 prime HS1 inverted HUDEP2 clone A2 H3K27ac-ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: About 2 million of HUDEP-2 cells upon 5 days of differentiation were harvested and fixed with 1% formaldehyde for 10 minutes at room temperature. Fixation was quenched with 0.125 M glycine. Cells were washed twice with ice-cold PBS, lysed in 0.13 mL of lysis buffer (10 mM Tris pH 8.0, 0.25% SDS, 2 mM EDTA, 1X protease inhibitors) and sonicated in a Covaris microtube with Covaris ultrasonicator (E220, Covaris). Sonicated chromatin was diluted with 0.2 mL of equilibration buffer (10 mM Tris pH 8.0, 233 mM NaCl, 1.66% Triton X-100, 0.166% sodium deoxycholate, 1 mM EDTA, 1X protease inhibitors) and spun down to pellet insoluble materials. Supernatant was mixed with 2 µg of antibody (H3K27ac, D5E4, Cell Signaling) and incubated overnight along with 10 µL of Dynabeads protein A (Thermo Fisher Scientific), which were washed twice with 0.1% BSA/PBS. After overnight rotating, beads were transferred to the tube containing chromatin and incubated for 2 hours. Beads were washed twice with RIPA-LS (10 mM Tris pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100), twice with RIPA-HS (10 mM Tris pH 8.0, 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100), twice with RIPA-LiCl (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate), and once with 10 mM Tris pH 8.0. After washing, 48 uL of elution buffer (10 mM Tris pH 8.0, 5 mM EDTA, 300 mM NaCl, 0.4% SDS) with 2 uL of 20 mg/mL of proteinase K (Viagen) were added to the beads and incubated for 1 hour at 55oC followed by overnight incubation at 65oC to decrosslink. Beads were magnetized and supernatant was purified with phenol-chloroform. DNA was precipitated with ice-cold absolute ethanol, washed with 75% ethanol, and eluted with 20 uL of 10 mM of Tris pH 8.0. Accel-NGS 2S Plus DNA library kit (21096, Swift Bioscience) was used to construct the library followed by 2S combinatorial dual indexing kit (28096, Swift Bioscience).
attributes.tag: GEO Accession
attributes.value: GSM4873123