GSM8878488: TRESLIN FLAG-IP with GINS4-mAID, Early-S, 5phIAA (IP); Homo sapiens; OTHER
Approximately 2×107 cells were harvested and lysed in 1 ml of IP lysis buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 5% glycerol, 3 mM MgCl2, 100 U benzonase (Millipore, #70746), complete protease inhibitor cocktail (Roche, #11873580001), and phosphatase inhibitor cocktail (Roche, #490684500)) at 10°C for 30 min. The lysate was centrifuged twice at 14,000 rpm for 15 min at 4°C to remove insoluble debris, followed by a pre-clearing step with 0.75 mg of Protein G Dynabeads (Invitrogen, #10004D) at 4°C for 1 h. Pre-cleared lysate was incubated with 1.5 µg of Protein G Dynabeads pre-bound with anti-FLAG M2 antibody (Sigma, #F1804) for 2 h at 4°C. Beads were washed twice with 1ml of IP wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 5% glycerol, complete protease inhibitor cocktail, and phosphatase inhibitor cocktail). Bound DNA was eluated twice by adding 90 µl of elution buffer (TE + 1% SDS) and subsequently by incubating at 45°C for 10 min. The combined eluate was treated with 20 µg RNase A for 1 h at 37°C, followed by treatment with 40 µg Proteinase K for 2 h at 50°C. DNA was purified using NucleoSpin Gel and PCR Clean-up Kit and quantified. NGS libraries were prepared from 2 ng of DNA using NEBNext Ultra II DNA Library Prep Kit for Illumina, and 50 bp paired-end sequencing was performed on the Illumina NovaSeq 6000 platform.