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Sample GSM7140996 Query DataSets for GSM7140996
Status Public on Jun 13, 2023
Title CH12_Repliseq_Rif1KO_shMcm6_early_rep1
Sample type SRA
Source name CH12
Organism Mus musculus
Characteristics cell type: CH12
sample group: Rif1KO_shMcm6_early
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
Data processing library strategy: Repli-seq
All RepliSeq analysis was wrapped into the reproducible Nextflow workflow repliseq-nf available at 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.
BioSample SAMN34060574
SRA SRX19857780

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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