title: GSM6756898: GC, plus 4-OHT, +NU-7441, + PFM01, 1 hr only, +antibody; Homo sapiens; ChIP-Seq
library_construction_protocol: GLASS-ChIP assay   Human U2OS cells with inducible AsiSI15 were grown to 50-60% confluency in 150 mm dishes and treated with 600 nM 4-OHT for 4 h at 37°C. When used, 10 µM NU7441 was added 30 min. prior to 4-OHT addition. After 4-OHT treatment, cells were fixed with 1% formaldehyde for 7 min at RT with gentle rotation. Crosslinking was stopped by addition of 125 mM glycine for 5 min, washed twice with cold PBS, harvested, flash frozen in liquid nitrogen and stored at -80°C.   For the GLASS-ChIP assay, we modified a standard protocol (Abcam) with a gentle lysis procedure and minimal, low-level sonication to rupture cells without extensive DNA damage as previously described16. The formaldehyde fixed cells were thawed at RT for 5 min, resuspended in RIPA buffer (50 mM Tris-HCl pH8.0, 150 mM NaCl, 2 mM EDTA pH8.0, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS) with 1x protease inhibitors (Pierce #A32955) and sonicated using a Cell Ruptor at low power, for 10 sec followed by 10 pulses after a 20 sec interval. Cell lysates were then centrifuged at 3000 rpm for 3 min at RT to remove the bulk of chromatin. The supernatant was then incubated with 1.6 µg of anti-DNA-PKcs pS2056 antibodies (abcam 124918) overnight at 4°C, followed by incubation with 25 µl Protein A/G magnetic beads (Pierce) at RT for 2 h. Beads were then washed sequentially  once in low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), once in high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), once in LiCl wash buffer (0.25 M LiCl, 1% NP-40, 1% Sodium Deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0).  Beads were then resuspended in TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA) and transferred to a fresh tube and finally eluted with 100 µl elution buffer (1% SDS, 100mM NaHCO3). Crosslinks were reverted for the elutions (65°C for 24 h) and DNA was purified with a Qiagen Nucleotide Clean up kit. DNA fragments <300 bp from ChIP elutions were separated by pulling down larger fragments using paramagnetic Ampure XP beads: 65 µl of Ampure XP beads were added to the uncrosslinked ChIP elution and mixed thoroughly. After 10 min incubation at RT, beads were isolated using a magnet. The supernatant was collected and 25 µl of fresh Ampure XP beads were added and mixed thoroughly. After 10 min at RT, beads were separated and the supernatant was purified using Qiagen Nucleotide Clean up kit. At the final step DNA was eluted in 60 µl of elution buffer (TE). To monitor the Mre11 nuclease dependence, we treated the human U2OS cells expressing inducible AsiSI with 10 µM NU7441, 100 µM PFM01 or PFM39 and 600 nM 4-OHT simultaneously for 1 hour at 37°C before harvesting cells.   Pellet ChIP  For DNA-PKcs pellet ChIP, the protocol for GLASS-ChIP was followed through the step where chromatin is isolated. The chromatin fraction was then resuspended in 2.2 ml RIPA buffer and was fragmented using a Diagenode Bioruptor on high setting for 30 min. with 10 sec on, 10 sec off. After removal of debris by centrifuging at 800 g for 3 min., the supernatant was incubated with 1.6 µg of anti-DNA-PKcs pS2056 antibodies (abcam 124918) overnight at 4°C, followed by incubation with 25 µl Protein A/G magnetic beads (Pierce) at RT for 2 h. The rest of the procedure was identical to GLASS-ChIP above except that the AMPure bead size selection was not performed. For DNA-PKcs GLASS-ChIP and pellet-ChIP  sequencing libraries, the eluted DNA was used to make sequencing libraries using the NEBNext Ultra or Ultra II DNA Library Prep Kit for Illumina (NEB) with NEBNext Multiplex dual index primers.