GSM9126411: Huh7 cell line, FLAG tag knock-in, infected with lenti-VPS72-FLAG virus, FLAG (ChIP); Homo sapiens; ChIP-Seq
When the cells reached 90–100% confluency, they were washed twice with PBS and treated with DMEM containing 1% formaldehyde, followed by a 5 min incubation on a shaker. After this step, 900 µL of 2.5 M glycine was added, and the cells were incubated for an additional 5 min on a shaker. The cells were then washed twice with PBS, scraped off, and collected by centrifugation at 500 g for 5 min at 4 °C. They were resuspended in lysis buffer (85 mM KCl, 0.5% CA-630, 5 mM PIPES pH 8, 1 mM PMSF, and Pierce Protease and Phosphatase Inhibitor from Thermo) and sonicated for 150 s using the M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA) at a peak power of 75 W, duty factor of 2%, 200 cycles/burst, and temperature maintained at 4 °C. Following sonication, the lysate was centrifuged at 1000 g for 5 min at 4 °C. The nuclear pellet was then resuspended in shearing buffer (10 mM Tris-HCl pH 8, 0.1% SDS, 1 mM EDTA pH 8, 1 mM PMSF, and Protease and Phosphatase Inhibitor) and subjected to sonication for 1560 s under the same conditions as before. After another centrifugation at 10,000 g for 10 min at 4 °C, the supernatant was collected. This supernatant was either stored as input or mixed with antibodies and incubated overnight on a shaker at 4 °C. After incubation, pre-blocked ChIP-grade Protein A/G Magnetic Beads (Thermo) were added and incubated on a shaker at 4 °C for 4 h. After this step, the beads were precipitated, and the supernatant was discarded. The beads were washed three times with sequential wash buffers: wash buffer I (150 mM NaCl, 20 mM Tris-HCl pH 8, 2 mM EDTA pH 8, 0.1% SDS, 1% Triton), wash buffer II (500 mM NaCl, 20 mM Tris-HCl pH 8, 2 mM EDTA pH 8, 0.1% SDS, 1% Triton), and wash buffer III (250 mM LiCl, 10 mM Tris-HCl pH 8, 1 mM EDTA pH 8, 1% sodium deoxycholate, 1% CA-630). The proteins were then eluted using elution buffer (1% SDS, 100 mM NaHCO3). All samples received a 5 M NaCl solution to achieve a final concentration of 0.2 M and were incubated overnight at 65 °C. After this incubation, the samples were treated with RNase A and Proteinase K, purified using the Monarch PCR & DNA Cleanup Kit (NEB) DNA libraries were prepared by using NEBNext Ultra II DNA Library Prep Kit for Illu minaandlibrarysizewasselectedbyusingPromegaProNexSize-Selective Purification System.