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| Status |
Public on Dec 13, 2017 |
| Title |
K562_A1_gH2Ax |
| Sample type |
SRA |
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| Source name |
K562
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| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
treatment: Aphidicolin
antibody: α-phospho-H2AX (S139) (Millipore 05-636)
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| Treatment protocol |
Plasmid transfections were performed using Lipofectamine 2000 transfection reagent (Life Technologies), cells were analyzed 24 h to 48 h post transfection. Lentiviral infection was carried out by spin infection (2250 rpm, 90 min, Beckman-Coulter Allegra X-12R centrifuge) with 8 µg/ml polybrene (Sigma), cells were incubated overnight prior to virus removal and selection with puromycin (1-2 µg/ml). Individual MISSION shRNA-expressing lentiviral vectors were from Sigma (Table S2). SiRNAs (Dharmacon ON-TARGET, Table S2) were transfected using DF-1 reagent following the manufacturer’s instructions (Dharmacon) and analyzed 48 h - 96 h post transfection.
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| Growth protocol |
K562 cells (American Type Culture Collection, ATCC) were cultured in RPMI-1640 (Gibco) supplemented with 10% BCS (Hyclone), BJ (ATCC) and IMR90 fibroblasts (Coriell) were cultured in MEM (Gibco), supplemented with 10% FBS (Gemini), 2 mM L-Glutamine (Gibco), 1 mM sodium pyruvate (Sigma), 10 mM Non-Essential Amino Acids (NEAA, Gibco), with or without 1% penicillin-streptomycin (Gibco). HCT116 H2AX KO cell lines (gift from W. Bonner) were cultured in DMEM (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin. U2OS cells with stably integrated LacO arrays (gift from T. Misteli) were cultured in DMEM supplemented with 10% FBS and 200 µg/ml hygromycin. Cells were maintained at 37 °C in a humidified incubator containing 5% CO2. Cell lines were negative for mycoplasma.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Sequencing libraries were constructed from DNA samples with the Illumina TruSeq V3 library construction protocol.
Cells were treated as indicated, crosslinked with 1% formaldehyde in PBS for 10 min, followed by quenching with 125 mM glycine. Cells were then resuspended in lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% nonidet P-40) to isolate nuclei. Nuclei were resuspended in micrococcal nuclease (MNase) digestion buffer (10 mM Tris pH 7.4, 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine) and 1.2 U/µl MNase was added for 30-45 min at 37 °C. The reaction was stopped by adding 50 mM EDTA and nuclear pellets were resuspended in 10mM Tris-HCl (pH8.0), 100 mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine. Lysates were sonicated briefly to disrupt nuclear membranes using an ultra sonicator water bath (Bioruptor, Diagenode). Diluted lysates were incubated o/n at 4 °C with the indicated antibodies after addition of 1% Triton X-100. IPs were performed using 30 µl Protein A/G magnetic beads (Pierce). Eluted DNA was purified with QIAquick PCR purification (Qiagen) according to the manufacturer instructions. Purified ChIP DNA was analyzed by qPCR using a LightCycler 480 II (Roche).
Sequencing libraries were constructed from DNA samples (ChIP and control samples) with the Illumina TruSeq V3 library construction protocol (Illumina, San Diego, USA).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Reads were demultiplexing stage by the bacl2fastq2 software (v2.15, Illumina) and trimmed for adapter and low quality squence with the Trimmotative software
ChIP-Seq Reads were mapped to the human genome with Bowtie2 v.2.1.0 (Langmead & Salzberg, 2012) with default parameters.
Peak calling was performed with SICER (v1.1) (Zang et al., 2009), with a window size of 200bp and a gap size of 600bp. For each ChIP sample, peak calling was performed relative to the respective input sample. Bigwig format signal tracks were generated with deeptools (Ramirez et al., 2016), normalizing for sequencing depth.
Genome_build: hg19
Supplementary_files_format_and_content: BED files contain peaks called
Supplementary_files_format_and_content: BW files contain depth-normalized read counts
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| Submission date |
Oct 11, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
David Sturgill |
| E-mail(s) |
[email protected]
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| Phone |
240-760-6725
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| Organization name |
NIH
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| Department |
NCI
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| Lab |
LRBGE
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| Street address |
Building 41, Rm B622
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| City |
Bethesda |
| State/province |
MARYLAND |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE104800 |
Replication stress shapes a protective chromatin environment across fragile genomic regions |
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| Relations |
| BioSample |
SAMN07770209 |
| SRA |
SRX3268809 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2808031_K562-A1-gH2Ax-W200-G600-FDR0.01-island.bed.gz |
233.3 Kb |
(ftp)(http) |
BED |
| GSM2808031_K562-A1-gH2Ax_rpkm.bw |
195.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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