|
| Status |
Public on Jun 01, 2021 |
| Title |
U2OS CDT1OE doxcycline Replicate 2 (Nascent strands) |
| Sample type |
SRA |
| |
|
| Source name |
Osteosarcoma
|
| Organism |
Homo sapiens |
| Characteristics |
strain: U2OS
cell type: Cultured cancer cell line
chip antibody: none
molecule subtype: Nascent DNA
|
| Treatment protocol |
Cells were treated with MLN4924 or doxyclcline to induce re-replication.
|
| Growth protocol |
Cancer cells were grown in DMEM medium, 10% FBS
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA from cell cultures was isolated, size fractionated and subject to exunuclease digestion as described in Aladjem et al., Science 281, 1005, 1998. and Wang et al., Molecular and Cellular Biology 24:3373-86, 2004. For ChipSeq, DNA molecules were enriched using antibodies against proteins of interest. Re-replicated DNA was isolated by by Brdu-cesium chloride gradient
Enriched ChIPed DNA molecules were sequenced by Illumina NextSeq 75 cycle High Output kit with single ennd of 75bp. For nascent DNA, re-replicated DNA, paired-end libraries of genomic DNA (gDNA) were prepared using Illumina TruSeq Nano DNA Library Prep kits. Control genomic DNA was fragmented to ~ 400 bp insert size on the Covaris which generates dsDNA fragments with 3' or 5' overhangs. The sheared DNA was blunt-ended and library size selection was done using sample purification beads. A single 'A' nucleotide was added to the 3' ends of the blunt fragments to prevent them from ligating to each other during the adapter ligation reaction. A corresponding single 'T' nucleotide on the 3' end of the adapter provided a complementary overhang for ligating the adapter to the fragment. The indexed adapters were ligated to the ends of the DNA fragments and then PCR-amplified to enrich for fragments that have adapters on both ends. The final purified product was then quantitated by qPCR before cluster generation and sequencing.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Unsynchronized doxycycline-treated U2OS nascent strand DNA
U2OS CDT1OE doxcycline NS-2
|
| Data processing |
Library strategy: NS-seq
For ChIP-Seq, peaks were called using the MACS algorithms againt input. Default parameters were used for sample calling.
For nascent strand peak calling the MACS was used. Nascent strand reads were called by comparison to genomic DNA. Peaks were filtered using the “peak-score” MACS2 metric in R (version 3.5.1) by accepting regions above the inflection-point threshold of “peak-scores” from the raw output.
Single read 75bp (NextSeq for ChipSeq) or Pair End 101, 125 and 150 bp (HiSeq or NovaSeq) for NGS sequencing
Genome_build: hg19
Supplementary_files_format_and_content: Bed file format of replication initiation peaks and ChipSeq
|
| |
|
| Submission date |
Apr 20, 2021 |
| Last update date |
Jun 01, 2021 |
| Contact name |
Christophe E Redon |
| E-mail(s) |
redonc@mail.nih.gov
|
| Phone |
240-760-7338
|
| Organization name |
NIH
|
| Street address |
37 Convent Drive
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE172417 |
DYNAMICS OF REPLICATION ORIGIN OVER-ACTIVATION |
|
| Relations |
| BioSample |
SAMN18810543 |
| SRA |
SRX10644225 |
