 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 31, 2019 |
| Title |
K562 treated with Etoposide for 36 hours bioreplica 1 |
| Sample type |
SRA |
| |
|
| Source name |
K562
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
cell type: chronic myelogenous leukemia cell line
|
| Treatment protocol |
For drug treatments, 1-2 million cells were seeded at 1 million cells per ml of medium per well in 24- (1ml media) or 6-well (2-3 ml) plates. After 16 hours, the following chemicals were added separately: 0.1% DMSO, 0.5mM H2O2, 1μM imatinib (Abmole Bioscience Inc, M3241), 0.0462µM romidepsin (Abmole Bioscience Inc, M2007), 1μM talazoparib (Abmole Bioscience Inc, M1732), 1μM SN-38 (Abmole Bioscience Inc, M3016), 40μM 10074-G5 (Sigma, 475957), 1μM YM-155 (Abmole Bioscience Inc, M2342) and 100μM etoposide (Abmole Bioscience Inc, M2326) and incubated for 6, 12, 24, 36 or 48 hours. All drugs (with exception of H2O2) were dissolved in DMSO, concentration of which was kept at 0.1% in all treatments. All treatments were done in two biological replicas for SSiNGLe-ILM. The following treatments were performed on separate batch of cells for SSiNGLe-SMS: DMSO, etoposide, SN-38, romidepsin, imatinib and talazoparib for 6, 12, 24 (with the exception of imatinib), 36 and 48 hours. For the Nt.BbvCI experiments, hydrogen peroxide treatment was done only for 6 hours and un-treated cells grown under the same conditions were used as controls.
|
| Growth protocol |
Human chronic myeloid leukemia cell line K562 was obtained from Cell Bank of Chinese Academy of Sciences. Cells were cultured in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Thermo Fisher Scientific) and 1% (v/v) pen-strep (Thermo Fisher Scientific) at 37°C and 5% CO2. Mouse neuroblastoma (N2a) and human cervical carcinoma HeLa cell lines were obtained from National Infrastructure of Cell Line Resource. N2a cells were cultured in MEM/EBSS (HyClone), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Thermo Fisher Scientific) and 1% (v/v) pen-strep (Thermo Fisher Scientific) at 37°C and 5% CO2. HeLa cells were cultured following at the same condition as K562 cells.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with formaldehyde in vivo, fragmented with MNase and used for genomic DNA extraction.
Samples were prepared according to the SSiNGLe-ILM protocol. Briefly, nuclei were isolated from cells crosslinked in vivo with formaldehyde and subjected to MNase treatment to fragment DNA to 150-500bp. The genomic DNA was then isolated after cross-link reversal and subjected to polyA-tailing using terminal transferase to tag positions of endogenous single-strand DNA breaks (products of MNase fragmentation would not be tailed under these conditions). The polyA-tailed single-strand DNA molecules were then used as templates for 2nd strand DNA synthesis with a chimeric 5’-DNA-RNA-3’ oligonucleotide followed by polyC-tailing of the 2nd strand DNA. The resulting polyA- and polyC-tailed double-strand DNA molecules were used for Illumina sequencing library construction using PCR with oligonucleotides containing appropriate adaptors at their 5’ ends.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
Library strategy: SSiNGLe-ILM
Only paired-end raw reads where read 1 started with 10G’s, read 2 started with 12T’s and each base of each read had Phred quality score >20 were selected.
Such reads were then aligned to the GRCh37/hg19 or GRCm38/mm10 assemblies of human or mouse genomes respectively using BWA-MEM (Version 0.7.12) with default settings. Only pairs of reads where both reads 1 and 2 uniquely mapped to the genome with appropriate configuration and spacing were kept.
Pairs of reads where fraction of T’s in the 20-base 5’ upstream sequence in the read 2 alignment was >40% were removed from the downstream analyses.
A single-strand break was defined as the first base after 12 T’s in the read 2.
Genome_build: hg19 for human and mm10 for mouse
Supplementary_files_format_and_content: bed.gz files containing coordinates of single-strand breaks
|
| |
|
| Submission date |
Oct 17, 2019 |
| Last update date |
Dec 31, 2019 |
| Contact name |
Huifen Cao |
| E-mail(s) |
hfcao_bnu@163.com, hfcao@hqu.edu.cn
|
| Phone |
+86 05926167250
|
| Organization name |
Huaqiao University
|
| Department |
School of Biomedical Sciences
|
| Lab |
Institute of Genomics
|
| Street address |
668 Jimei Road
|
| City |
Xiamen |
| State/province |
Fujian |
| ZIP/Postal code |
361021 |
| Country |
China |
| |
|
| Platform ID |
GPL20795 |
| Series (1) |
| GSE139011 |
Novel approach reveals genomic landscapes of single-strand DNA breaks with nucleotide resolution in human cells |
|
| Relations |
| BioSample |
SAMN13047959 |
| SRA |
SRX7011501 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4126131_Etoposide_36h.B1.without.polyA.untailing.bed.gz |
6.0 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
