GSM8912044: DLD-1, NELFC-AID, replicate 1, BrdU-IP; Homo sapiens; OTHER
Genomic DNA was extracted using the Blood & Cell Culture DNA Midi Kit with 100/G Genomic-tips (Qiagen). After measuring DNA concentration, 10 µg of DNA was dissolved in 200 µL of TE buffer and subjected to sonication using ultrasound (30 seconds on, 30 seconds off, for 60 cycles) with the Bioruptor II (BMbio). A 15 µL aliquot of the sonicated DNA solution was set aside as the input sample, and the remaining 150 µL was heated at 96°C for 10 min and snap-cooled in ice-cold water to denature the DNA before immunoprecipitation with an anti-BrdU antibody (Clone B44, BD Biosciences).To the denatured DNA solution, an equal volume of 2× BrdU-IP buffer (200 mM sodium phosphate, pH 7.3, 280 mM NaCl, 0.1% Triton X-100) was added. Next, 20 µL of BrdU antibody (3.75 µg) pre-bound to Protein G Dynabeads (Life Technologies) was added to the solution and incubated at 37°C for 1h. The tube containing the solution was placed on a magnetic stand, and the beads were washed twice with 1×BrdU-IP buffer and once with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Bead-bound DNA was eluted in 50 µL of elution buffer (50 mM Tris-HCl, pH 8.0, 1% SDS). Both the input DNA and BrdU-IP DNA were purified using AMPure beads (Beckman Coulter) following the manufacturer's instructions and dissolved in 100 µL of 1 mM Tris-HCl (pH 8.0). A total of 4 ng of immunoprecipitated single-stranded DNA (ssDNA) fragments in 30 μl of 1 mM Tris-HCl (pH 8.0) was mixed with 5 μl of 3 mg/ml custom-synthesized 8N random primer, heated at 95°C, and immediately snap-cooled on ice. The samples were then incubated with 2.5 μl (12.5 units) of Klenow fragment (NEB) and 5 μl of 2 mM dNTPs in a total volume of 50 μl to convert single-stranded DNA (ssDNA) into double-stranded DNA (dsDNA). The resulting dsDNA was purified using AMPure XP beads (Beckman Coulter), and its concentration was determined by fluorometric quantification (PicoGreen; Life Technologies). Fragment size distribution was analyzed with an Agilent TapeStation. Subsequently, all purified dsDNA was used for Illumina library preparation with the NEBNext Ultra II DNA Library Prep Kit (NEB).