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Status |
Public on Sep 18, 2023 |
Title |
GC, no 4-OHT, +NU-7441, +antibody, rep1 |
Sample type |
SRA |
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Source name |
U2OS
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Organism |
Homo sapiens |
Characteristics |
cell line: U2OS cell type: osteosarcoma chip antibody: phospho-DNA-PKcs(S2056)(Abcam ab124918) treatment: no 4-OHT, +NU7441, +antibody
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Extracted molecule |
genomic DNA |
Extraction 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. DNA-PKcs GLASS-ChIP and pellet-ChIP: NovaSeq SP platform with PE150
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
All processed data files are Hg19 except files with 'light' in the name which are Hg38
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Data processing |
DNA-PKcs GLASS-ChIP and pellet-ChIP :Raw data was pre-processed with BedTools fastp, mapped with BWA-MEM, duplicates were removed with SAMTools RmDup, and peaks called with MACS2. AsiSI-associated data was processed using genome build Hg19; Cas9-associated data was processed using genome build Hg38. Mre11 ChIP data: For Glass-ChIP, no-antibody sample libraries were used as controls whereas for pellet-ChIP, input DNA libraries were used as controls. body sample libraries were used as controls whereas for pellet-ChIP, input DNA libraries were used as controls. Bedgraph output files from MACS2 were converted to bigwig files using ucsc-wigtobigwig (Version 357) Assembly: all processed data files are Hg19 except files with 'light' in the name which are Hg38
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Submission date |
Nov 22, 2022 |
Last update date |
Sep 18, 2023 |
Contact name |
Tanya T. Paull |
E-mail(s) |
tpaull@utexas.edu
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Phone |
5122327803
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Organization name |
Univ. of Texas at Austin
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Department |
Molecular Biosciences
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Lab |
Paull
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Street address |
2500 Speedway MBB 2.448
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City |
Austin |
State/province |
TX |
ZIP/Postal code |
78712 |
Country |
USA |
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Platform ID |
GPL24676 |
Series (1) |
GSE218590 |
Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice |
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Relations |
BioSample |
SAMN31841116 |
SRA |
SRX18353251 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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