GSM2585718: GM12878_CPD_20J_cell_B; Homo sapiens; OTHER
Genomic DNA were extracted by PureLink Genomic DNA kit (Thermo). Genomic DNA was sheared by sonication with Q800 Sonicator (Qsonica) to generate fragments averaging 600 bp in length and then purified by an equal volume of HighPrep PCR beads (MagBio). DNA fragments were eluted by 0.1xTE and the concentration was determined by Qubit (Thermo). Purified DNA fragments (1 µg) were used for end-repair by NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) following the manufacturer's instruction. Ad1 (100 pmol) was ligated to both ends at 16°C overnight, then purified by HighPrep PCR beads (60 µl) and eluted in 55 µL of 0.1x TE. Damaged DNA immunoprecipitation was performed as described previously with modification. DNA were denatured by adding 20 µL of 8M urea, boiling for 1 min and immediately putting in ice water for 1 min. Then 20 µg of denatured sonicated salmon sperm DNA (Thermo) and 11 µL 8x IP Buffer (1x IP Buffer: 20 mM Tris-Cl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, and 0.5% sodium deoxycholate) were added, followed by incubation with antibody-coated beads which were prepared as described previously (4) at 4°C overnight.  The beads were then washed sequentially with Wash Buffer U (20 mM Tris-Cl pH 8.0, 2 mM EDTA, 1% Triton X-100, and 2M Urea), Wash Buffer II (20 mM Tris-Cl pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, and 0.1% SDS), Wash Buffer III (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 150 mM LiCl, 1% NP-40, and 1% sodium deoxycholate), Wash Buffer IV (100 mM Tris-Cl pH 8.0, 1 mM EDTA, 500 mM LiCl, 1% NP-40, and 1% sodium deoxycholate) and TE (10 mM Tris-Cl pH 8.0 and 1 mM EDTA) at room temperature. The fragments containing damage were eluted by incubation with 100 µL of Elution Buffer (50 mM NaHCO3, 1% SDS) at 65°C for 10 min on a thermomixer at 1100 rpm. The eluted DNA was then isolated by phenol/chloroform extraction followed by ethanol precipitation.  Primer Bio3U was attached to IP purified DNA and extended by NEBNext Q5 DNA polymerase, followed by ExoI (New England Biolabs) treatment and  HighPrep PCR beads cleanup, then denatured and captured by Dynabeads MyOne Streptavidin C1 (Thermo) as described previously (2). After incubation at 4°C for 1 hr, the beads were washed by 1x B&W buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl) and 1x TE, followed by incubation with 2 µL of USER Enzyme (New England Biolabs) in 18 µL of 0.1x TE at 37°C on a thermomixer at 900rpm for 25 min. The supernatant was separated by the magnetic stand and transferred to a new tube. Then the beads were washed with 25 µL of 1x B&W Buffer and the supernatant was collected and combined with supernatant from the last step. For subtractive hybridization, 20 pmol of oligo SH were added, followed by boiling for 1 min and naturally cooling down to room temperature. Oligo SH was then captured by incubating with 10 µL of Dynabeads MyOne Streptavidin C1 for 1 hr at room temperature. After separating the liquid on a magnet and transferring supernatant to a new tube, the beads were washed with 35 µL of 1x B&W Buffer and the supernatant was transferred to the same tube. DNA were purified by phenol/chloroform extraction and ethanol precipitation.  The following ligation of Ad2 and PCR amplification were performed as described previously (2). Libraries were sequenced from both ends on Hiseq 2500 by the University of North Carolina High-Throughput Sequencing Facility.
GEO Accession
GSM2585718