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| Status |
Public on Oct 26, 2021 |
| Title |
MCF7_SCL-exo_EV_EtOH_Rep3 |
| Sample type |
SRA |
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| Source name |
EV cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: MCF-7-derived breast cancer cells
genotype/variation: EV
treatment: 0.1% EtOH for 50 minutes
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| Treatment protocol |
For RNA-seq, ChIP-seq and SCL-exo-seq, cells were kept for 48h in phenol-red-free DMEM (Gibco 31052) supplemented with 2.5% dextran-charcoal-treated fetal calf serum (Eurobio S116365181W), glutamine, sodium pyruvate, non-essential amino acids, Penicillin-Streptomycin and Geneticin at 37°C and 5% CO2 before treatment with 10 nM estradiol or 0.1% EtOH.
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| Growth protocol |
Cells were grown in high glucose and pyruvate containing DMEM (Gibco 41966) supplemented with 10% fetal calf serum (Eurobio S116365181H), non-essential amino acids (Gibco 11140035), Penicillin-Streptomycin (Gibco 15240) and Geneticin (Gibco 11811064) at 37°C and 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq assays, cells were fixed in 1.5% formaldehyde (Sigma F8775) for 10 min at room temperature, the reaction was stopped by the addition of glycine (100 mM). Cells were lysed in lysis buffer (150 mM Tris-HCl pH 8.1, 10 mM EDTA, 0.5% Empigen BB, 1% SDS, protease inhibitor cocktail (Roche 5056489) and sonicated using a bioruptor (Diagenode, 15 min 30 sec ON /30 sec OFF). Sonicated chromatin was incubated at 4°C overnight with antibodies (ERa: mix of TE111.5D11 from Abcam and HC-20 from Santa Cruz Biotechnology, H3K4me3: 04-745 from Millipore, H3K27me3: 07-449 from Millipore) in IP buffer (2.8 ng/mL yeast tRNA, 20 mM Tris-HCl, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, proteases inhibitor cocktail). Complexes were recovered after 4h incubation with 45 mL protein A- and 15 mL protein G-conjugated sepharose beads slurry at 4°C. Beads were washed in washing buffers WB1 (20 mM Tris-HCl pH 8, 2 mM EDTA, 150 mM NaCl, 0.1% SDS, 1 % Triton X-100), WB2 (20 mM Tris-HCl pH 8, 2 mM EDTA, 500 mM NaCl, 0.1% SDS, 1 % Triton X-100), WB3 (10 mM Tris-HCl pH 8, 1 mM EDTA, 250 mM LiCl, 1 % deoxycholate, 1 % NP-40), WB4 (10 mM Tris-HCl pH 8, 1mM EDTA) and DNA fragments were eluted in extraction buffer (1% SDS, 0.1 M NaHCO3). For RNA-seq, total RNAs were extracted with the RNeasy Plus kit from QIAGEN including a DNase digestion step, and mRNA were enriched by selection with poly-dT beads. For SCL-exo-seq, genomic DNA was extracted using reagents from the DNeasy Blood and Tissue kit (QIAGEN).
ChIP-seq libraries (2 replicates) were generated with DNA fragments from 6 to 9 pooled individual ChIP experiments and the TruSeq kit from Illumina according to the manufacturer's protocol. For RNA-seq, three replicate libraries of each condition (TruSeq stranded mRNA, Illumina) were prepared and sequenced (single reads of 75 bases) at the Genomic Paris Center facility (Paris, France). For each SCL-exo experiment, 8 micrograms of gDNA were sonicated two times 7 min (30 sec ON /30 sec OFF) and two times 14 min (30 sec ON /30 sec OFF) with a bioruptor (Diagenode). Glucosylation and biotinylation of 5hmC were performed with the Hydroxymethyl Collector kit (Active Motif), followed by on beads-exonuclease digestion of the captured fragments and library preparation (TrueSeq library preparation kit, Ilumina). For normalization purpose, 400 pg of 5hmC Control DNA provided by the Hydroxymethyl Collector kit were added to each sample as spike-in. Libraries from 3 independent SCL-exo experiments were sequenced with a HiSeq 1500 (Illumina) by the GEH facility (Rennes, France).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Library strategy: SCL-exo-seq
All ChIP-seq and SCL-exo-seq sequencing reads were mapped to hg19 with using Bowtie (Langmead et al., Genome Biol. 2009). SAMtools (Li et al., Bioinformatics 2009) generated bam files which were processed with MACS (Zhang et al., Genome Biol. 2008) to generate wig files. Peak calling followed the procedure descibed in Sérandour et al (Nucleic Acids Res., 2012).
RNA-seq reads were mapped to hg19 with Bowtie (Langmead et al., Genome Biol. 2009) and transcripts were quantified with RSEM (Li and Dewey, Bioinformatics 2011). Differentially expressed genes were identified from RNA-seq data by the R package DESeq2 (Love et al., Genome Biol. 2014) after filtering of the raw data using the R package HTSFilter (Rau et al., Bioinformatics 2013).
SCL-exo-seq reads were treated as descibed (Sérandour et al., Bio-Protoc. 2018) using the python script available at https://mycore.core-cloud.net/index.php/s/4gyZ9dLTqgo86dt on SAM files.
Genome_build: hg19
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| Submission date |
Apr 26, 2021 |
| Last update date |
Oct 26, 2021 |
| Contact name |
Gilles Salbert |
| Organization name |
University Rennes 1
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| Department |
IGDR UMR6290
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| Street address |
263 Av. Gal. Leclerc
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| City |
Rennes |
| ZIP/Postal code |
35200 |
| Country |
France |
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| Platform ID |
GPL18480 |
| Series (1) |
| GSE173344 |
TET2-mediated deprogramming of breast cancer cells |
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| Relations |
| BioSample |
SAMN18872548 |
| SRA |
SRX10681705 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5266221_SCL-exo_EV_EtOH_Rep3.wig.gz |
16.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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