GSM7031960: H3K27ac hiChIP from hASCs of pear shaped Subject 2 abdominal depot; Homo sapiens; OTHER
Briefly, approximately 5x106 cells were crosslinked in 1% (methanol-free, dissolved in phosphate buffered saline - PBS) formaldehyde for 10 minutes at room temperature in 10ml final volume. Formaldehyde was quenched with the additional of 1.5ml 1M Glycine for 5 minutes at room temperature. Cells were scraped and lysed in lysis buffer (1% Triton x-100, 0.1% SDS, 150 mM NaCl, 1mM EDTA, and 20 mM Tris, pH 8.0). Cells were rotated in a cold room in lysis buffer for 1 hour to isolate nuclei. Nuclei were pelleted by centrifugation and resuspended in 100ul 0.5% SDS and incubated for 10 minutes at 65C. SDS was quenched by the addition of Triton-X and incubated for 15 minutes at 37°C. Nuclei were incubated overnight at 37C in a vigorous shaker (speed – 850rpm) in the presence of MboI (375U). Mbo I was heat inactivated at 65 for 20 minutes and samples were incubated at room temperature for 20 minutes to cool. Biotin fill-in of sticky ends were performed for 1 hour at 37C in a vigorous shaker (speed – 850rpm) followed by ligation of blunt ends at room temperature for 6 hours while rotating. Nuclei were spun and resuspended in lysis buffer in the presence of 5ug H3K27ac antibody (abcam cat), and incubated overnight on a rotator at 4C. Next day, antibody chromatin complexes were pulled down with protein A paramagnetic beads and were washed once in Wash Buffer 1 (1% Triton, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, and 0.1% NaDOC), twice in Wash Buffer 2 (1% Triton, 0.1% SDS, 500 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, and 0.1% NaDOC), once in Wash Buffer 3 (0.25 M LiCl, 0.5% NP-40, 1mM EDTA, 20 mM Tris, pH 8.0, 0.5% NaDOC), and finally, in TE-buffer. After the removal of TE-buffer, DNA was eluted from the beads in elution buffer. DNA was quantified with Qubit dsDNA HS kit. Approximately, 40-50ng DNA was used for biotin pull-down with streptavidin paramagnetic beads. Sequencing libraries were constructed with Nugen Ovation Ultralow V2 kit according to the manufacturer's recommendations. Libraries were quantified with Quibt dsDNA HS kit and subjected to bioanalyzer fragment analysis before paired-end sequencing