GSM8454548: untreated, WT, rep2; Homo sapiens; OTHER
Briefly, media was gently aspirated, and cells were lysed with MB buffer prewarmed at 55℃  (5.6 M guanidine thiocyanate 10 mM Tris–HCl (pH 6.5), 20 mM EDTA, 4% Triton X-100, 1% Sarkosyl, and 1% DTT). After scraping, the viscous lysate was passed through 1mL pipette tip six times, then transferred into 1.5 mL Eppendorf tube. The lysate was further sheared by passing it through a 19-gauge needle for six times, then equal volume of pre-chilled 100% ethanol was added to precipitate free and protein-linked DNA. After a full speed sedimentation at 4℃ for 20 min, the supernatant was carefully removed and the pellet was washed with a 50% ethanol wash buffer (20 mM Tris–HCl pH 6.5, 150 mM NaCl and 50% ethanol). The wash buffer was carefully aspirated and the remaining sedimented material was saved at -80℃ with no air-drying. The next day, precipitated DNA was resuspended in 1 mL 1% SDS buffer containing 20 mM Tris-HCl (pH 7.4), sheared by passing through a 21-guage needle six times, transferred to 1 mL Covaris milliTUBE, and subjected to ultrasonication for 12-14 minutes in a Covaris S-220 instrument following the manufacturer's protocol to generate DNA fragments in the 200-500bp size range. Following ultrasonication, KCl-SDS precipitation was used as previously described with some modifications. Briefly, 1M KCl and 1M Tris-HCl (pH 7.4) were added to a final concentration of 100 mM and 20mM; respectively, to precipitate protein-linked DNA. Samples were chilled on ice for 5 min then spun at full speed, 4℃, for 5 min. The precipitated protein-linked DNA was then washed with 1.5 mL of 100mM KCl buffer containing 20mM Tris-HCl (pH 7.4), heated at 55℃, chilled on ice, then spun again. The last wash was repeated one more time. The final pellet containing protein-linked DNA was then resuspended in a 100mM KCl buffer containing 20 mM Tris-HCl (pH 7.4), 10mM EDTA, and 0.2mg/ml proteinase K, and incubated overnight at 55℃ to digest DPCs and reverse the crosslink. The next day, samples were chilled on ice for 5 min, and supernatant was carefully collected after a full speed centrifuge at 4℃ for 10 min. Samples were then purified with Qiagen PCR purification column, and amount of purified DNA was measured with Qubit dsDNA High Sensitivity Assay kit following the manufacturer's guidelines (Fig. S2). Then, equal amounts of protein-linked DNA precipitated from each group were used to prepare DNA libraries for next-generation sequencing. DNA libraries were prepared using the NEBNext Ultra II DNA Prep Kit for Illumina following the manufacturer's protocol. Libraries with 200-500 bp were selected using the E-Gel Ex 2% agarose gel ran at 35v for 1.5 hour. Size-selected libraries were then purified using the MinElute Gel extraction kit and quantified with the Qubit. For library QC, libraries were diluted to 1 pM and amplified with kapa primers (kapa forward: AATGATACGGCGACCACCGA; kapa reverse: CAAGCAGAAGACGGCATACGA), where similar Ct values were expected. Then, libraries were pooled with equimolar ratio.