|
| Status |
Public on Sep 15, 2022 |
| Title |
CH12_C13_earlyS_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
CH12
|
| Organism |
Mus musculus |
| Characteristics |
cell line: CH12
clone: C13
cell cycle phase: earlyS
|
| Treatment protocol |
Two million asynchronously dividing cells were seeded and incubated with 100mM BrdU for 2h in a light protected environment to maintain BrdU stability.
|
| Growth protocol |
CH12 cells cultured in complete RPMI medium with IL4/CD40/TGF beta stimulation.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
BrdU labled DNA was extracted via phenol, phenol-chlorophorm extraction and subjected to immunoprecipitation
Repli-seq was performed as previously described (Marchal C, 2018). In brief, Cells were fixed and incubated with a mix of RNase A and propidium iodide . For each sample, three fractions were sorted: G1 phase, early S phase and late S phase cells, 50 000 cells per fraction Sorted cells were lysed . Extracted DNA was sonicated for 9 min in a Diagenode Bioruptor resulting in 100-500 bp DNA fragments . Sonicated DNA was subjected to end-repair and adapter ligation using the NEBNext® Ultra™ II DNA Library Prep Kit (NEB) following the NEB protocol. Adapter-ligated DNA was incubated with 25 mg/ml of anti-BrdU antibody (BD Pharmingen) for 4h with rotation followed by incubation with 40 μg of anti-mouse IgG antibody (Sigma) for 1h with rotation (light protected) which was followed by DNA purification
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Repli-Seq
CH12_RT_C13_loess.bw
|
| Data processing |
Library strategy: Repli-seq
Repli-seq
All RepliSeq analysis was wrapped into the reproducible Nextflow workflow repliseq-nf available at https://github.com/t-neumann/repliseq-nf loosely following the steps previously described in Marchal et al., 2018:
Raw reads from replicates of early (E) and late (L) fractions were trimmed with trim_galore v0.6.5 and aligned to the mouse mm9 reference sequence using bwa mem v0.7.17 and unmapped reads, secondary alignments and multimapping reads were filtered with samtools v1.9.
Replicates were merged using samtools and read duplicates were filtered using picardTools MarkDuplicates.
E/L log2 ratios were calculated in 5kb windows using deeptools bamCompare v3.4.1.
Loess smoothing was performed with bedtools v2.29.2 and a custom R script using the preprocessCore package v1.46.0 with a span size of 300 kb and bigwig tracks were produced using kent_tools v377.
The correlation between replicates was assessed with deepTools multiBamSummary.
Genome_build: NCBI mm9
Supplementary_files_format_and_content: bigWig of loess smoothed E/L log2 ratios.
|
| |
|
| Submission date |
Nov 19, 2020 |
| Last update date |
Sep 15, 2022 |
| Contact name |
Tobias Neumann |
| Organization name |
IMP
|
| Street address |
Campus-Vienna-Biocenter 1
|
| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE161819 |
DNA replication timing directly regulates the frequency of oncogenic chromosomal translocations [Repli-seq] |
| GSE161822 |
DNA replication timing directly regulates the frequency of oncogenic chromosomal translocations |
|
| Relations |
| BioSample |
SAMN16837343 |
| SRA |
SRX9532670 |
