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| Status |
Public on Sep 07, 2020 |
| Title |
S3 2x (βA-globin+ β-actin) |
| Sample type |
SRA |
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| Source name |
DT40 Chicken cell line
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| Organism |
Gallus gallus |
| Characteristics |
cell line: DT40
cell type: dervived from a Bursal lymphoma
genotype: 2x (betaA-globin+ beta-actin)
fraction: S3
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| Growth protocol |
DT40 cells were grown in RPMI 1640 Glutamax (Thermo Fisher Scientific #61870010) containing 10% FBS, 1% chicken serum, 0.1 mM β-mercaptoethanol, 200 U/mL penicillin, 200 µg/mL streptomycin and 1.75 µg/mL amphotericin B, at 37°C, under an atmosphere containing 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Exponentially growing cells were pulse-labeled with 5-Bromo-2’-deoxyuridine for 1h and four S-phase fractions (S1 to S4) were collected by flow cytometry or from an asynchronous cell population (AS). The cells were treated with lysis buffer (50 mM Tris pH 8.0; 10 mM EDTA pH 8.0; 300 mM NaCl; 0.5% SDS, 0.2 mg/ml of freshly added proteinase and 0.5 mg/ml of freshly added RNase A), incubated at 65°C for 2 h and stored at -20°C, in the dark. Genomic DNA was isolated from each sample by phenol-chloroform extraction and alcohol precipitation and sonicated four times for 30s each, at 30s intervals, in the high mode at 4°C in a Bioruptor water bath sonicator (Diagenode), to obtain fragments of 500 to 1000 bp in size. The sonicated DNA was denatured by incubation at 95°C for 5 minutes. We added monoclonal anti-BrdU antibody (BD Biosciences #347580) at a final concentration of 3.6 μg/ml in 1x IP buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 150 mM NaCl, 0.5% Triton X-100, and 7 mM NaOH). We used 30 μl or 50 µl of protein-G-coated magnetic beads (from Ademtech #4342 or Thermo Fisher Scientific #10004D, respectively) per sample to pull down the anti-BrdU antibody. Beads and BrdU-labeled nascent DNA were incubated for 2-3 hours at 4°C, on a rotating wheel. The beads were then washed once with 1x IP buffer, twice with wash buffer (20 mM M Tris pH 8.0, 2 mM EDTA pH 8.0, 250 mM NaCl, 0.25% Triton X-100) and then twice with 1x TE buffer pH 8.0. The DNA was eluted by incubating the beads at 37°C for 2 h in 250 µl 1x TE buffer pH 8.0, to which we added 1% SDS and 0.5 mg/ml proteinase K. DNA was purified by phenol-chloroform extraction and alcohol precipitation and resuspended in 50 µl TE. Immunoprecipitated NS were amplified by whole-genome amplification (GenomePlex Complete Whole Genome Amplification kit #WGA2; Sigma) according to the manufacturer’s recommendations to obtain sufficient DNA amount.
Libraries were constructed with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645S) following the manufacturer’s instructions with minor modifications. For the adaptor ligation, undiluted adaptor and no size selection were used. The library amplification was performed using NEB-Next Multiplex Oligos for Illumina (NEB #E7710S) with nine different NEB-Next index primer and the NEB-Next Universal PCR Primer (NEB #E6861A) with three PCR cycles. Library purification was performed with the SPRI-select Reagent kit (Beckman coulter #B23317), and the final elution step was reduced to 33µl of 0.1×TE. The mean size of the library molecules determined on an Agilent Bio-analyser High Sensitivity DNA chip (Agilent technologies, #5067–4626) was 330-350 bp. Sequencing was performed on a NextSeq 500 Illumina sequencer with a High Output 150 cycles flow cell (paired-end reads of 75 bp) according to standard procedures. For each sample, 15-20 M of reads were generated.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Nascent strands of DNA
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| Data processing |
Library strategy: Repli-Seq
Paired-end sequencing data were mapped on the galGal5 chicken genome using bowtie2 (Langmead & Salzberg, 2012). For each timing fraction, replication timing was computed using 50kb sliding windows at 10kb intervals, normalized by the global and local genomic coverage of the asynchonous cell population, to normalize for total and local coverage variations. Then the centered and standardized timing profiles were smoothed using cubic splines (smooth.spline function of R). In order to provide a single RT profile combining all fractions, we used the method proposed by (Du et al, 2019), by computing the weighted average WA = (0.917*G1) + (0.750*S1) + (0.583*S2) + (0.417*S3) + (0.250*S4) + (0*G2). An increase in WA indicates an earlier timing.
Genome_build: galGal5 chicken genome
Supplementary_files_format_and_content: Bed Graph files were generated
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| Submission date |
Jun 30, 2020 |
| Last update date |
Sep 08, 2020 |
| Contact name |
Caroline Brossas |
| E-mail(s) |
caroline.brossas@ijm.fr
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| Phone |
+33(1)57278124
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| Organization name |
Institut Jacques Monod, Université de Paris, CNRS UMR 7592
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| Lab |
Domaines chromatiniens et réplication
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| Street address |
15 rue Hélène Brion
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| City |
Paris |
| ZIP/Postal code |
75013 |
| Country |
France |
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| Platform ID |
GPL19787 |
| Series (1) |
| GSE153566 |
Clustering of strong replicators associated with active promoters is sufficient to establish an early-replicating domain |
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| Relations |
| BioSample |
SAMN15407350 |
| SRA |
SRX8641921 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4647409_timing_cl6_S3_gal5_chr1_normalized_centered.bed.gz |
2.6 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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