|
| Status |
Public on Jun 13, 2023 |
| Title |
priB_Repliseq_Rif1KO_Het_late_rep4 |
| Sample type |
SRA |
| |
|
| Source name |
priB
|
| Organism |
Mus musculus |
| Characteristics |
cell type: primary B cells
sample group: Rif1KO_Het_late
|
| 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 wit 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
Libraries were prepared following standard Illumina protocols
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 |
NextSeq 2000 |
| |
|
| Description |
Repli-Seq
priB_Repliseq_Rif1Het_RT_5kb_loess.bw
priB_Repliseq_Rif1Het_RT_20kb_loess.bw
priB_Repliseq_Rif1Het_RT_5kb_loess.bedGraph
priB_Repliseq_Rif1Het_RT_20kb_loess.bedGraph
|
| Data processing |
library strategy: 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 and 20kb 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.
RT domains were called with hmm_bigwigs v1.3
Assembly: NCBI mm9
Supplementary files format and content: bigWig of loess smoothed E/L log2 ratios.
Supplementary files format and content: bed file of hmm assigned RT domains
|
| |
|
| Submission date |
Apr 03, 2023 |
| Last update date |
Jun 13, 2023 |
| Contact name |
Tobias Neumann |
| Organization name |
IMP
|
| Street address |
Campus-Vienna-Biocenter 1
|
| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
| |
|
| Platform ID |
GPL30172 |
| Series (2) |
| GSE228878 |
RIF1 regulates replication origin activity and early replication timing in B cells [Repli-Seq] |
| GSE228880 |
RIF1 regulates early replication timing in murine B cells. |
|
| Relations |
| BioSample |
SAMN34060547 |
| SRA |
SRX19857775 |
