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| Status |
Public on Oct 01, 2019 |
| Title |
A1023C19: Input-untreated-2 |
| Sample type |
SRA |
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| Source name |
Basal breast cancer cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MDA-MB-468
tissue: Basal breast cancer cells
passages used: From 6 to 14
chip antibody: none (input)
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| Treatment protocol |
Cells were harvested by trypsinization. After adding media to stop trypsin, cells were centrifuged at 500 x g for 5 min at room temperature. The pelleted cells were resuspended in media, counted and crosslinked with 37% formaldehyde (final concentration 1%) for 10 min at room temperature. The crosslinking was stopped by addition of 2.5 M glycine (final concentration 0.125 M) for 5 min at room temperature followed by centrifugation at 500 x g for 5 min at 4°C.
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| Growth protocol |
MDA-MB-468 cells grown in 145 mm2 dishes until ~ 70% confluence were treated with EGF (100 ng mL-1) for 72h in biological triplicate and untreated cells were used as an appropriate control.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The pelleted cells were washed twice with ice-cold PBS1x by centrifuging at 500 x g for 5 min at 4°C. For lysis of crosslinked chromatin, the pellet was resuspended in lysis buffer A (50 mM Tris HCl pH8, 10 mM EDTA, 1% SDS, 50 x protease inhibitor cocktail) at room temperature and incubated for 5 min on rotating wheel. Next, lysate was centrifuged at 300 x g for 10 min at 8°C to prevent SDS precipitation and the supernatant was discarded. For chromatin shearing, the pellet was sheared in buffer B (25 mM Tris HCl pH8, 3 mM EDTA, 0.1% SDS, 1% Triton X-100, 150 mM NaCl, 50 x protease inhibitor cocktail) to approximatively 200-600 base-pair average size using the Bioruptor Pico (Diagenode). After centrifugation at 20 000 x g, 4ºC for 15 min, the supernatant containing sheared chromatin was used for immunoprecipitation. 25 µL (10%) of sheared chromatin was used as input DNA to normalize sequencing data. As supplement control for normalization, we used spike-in chromatin Drosophila and spike-in antibody (Active motif). For chromatin immunoprecipitation (1 million cells per ChIP), it was carried out using sheared chromatin and antibody H3K9me2 complexed to Dynal Protein G magnetic beads (Thermo Fisher). Briefly, 6 µL of H3K9me2 antibody was mixed with 1 µg of spike-in antibody. 23 µL of magnetic beads were washed three times in ice-cold buffer C (20 mM Tris HCl pH8, 2 mM EDTA, 0.1% SDS, 1% Triton X-100, 150 mM NaCl, 50 x protease inhibitor cocktail) and incubated with the mix of H3K9me2 and spike-in antibody for 4h at room temperature on rotating wheel in buffer C (494 µL). After the quick-spin and the removal of supernatant, the beads were resuspended in 100 µL buffer C. Each 50 µL of the latter was incubated with 250 µL of sheared chromatin mixed previously with 50 ng of spike-in chromatin Drosophila (250 µL chromatin of interest: 2,5 µL of spike-in chromatin, v:v) at 4°C on rotating wheel, overnight (~ 16h). After quick-spin, the supernatant was discarded and the beads were successively washed in buffer C, buffer D (20 mM Tris-HCl pH8, 2 mM EDTA, 0.1% SDS, 1% Triton X-100, 500 mM NaCl), buffer E (10 mM Tris-HCl pH8, 0.25 M LiCl, 0.5% NP-40, 0.5% Sodium Deoxycholate, 1 mM EDTA) and buffer F (10 mM Tris-HCl pH8, 1 mM EDTA, 50 mM NaCl). Finally, input and immunoprecipitated chromatin samples were resuspended in solution containing TE buffer/ 1% SDS, decrosslinked by heating at 65°C overnight and subjected to both RNase A and Proteinase K treatments.
Illumina compatible libraries were prepared from input and immunoprecipitated DNAs using the Illumina TruSeq ChIP library preparation kit according to the manufacturer’s protocol. Briefly, 4 to 10ng of DNA were subjected to subsequent steps of end-repair, dA-tailing and ligation of TruSeq indexed Illumina adapters. After a final PCR amplification step, the 24 resulting barcoded libraries were equimolarly pooled in 2 groups quantified using a qPCR method (KAPA library quantification kit, Roche) before sequencing on the Illumina HiSeq2500 instrument. Each pool was loaded on 1 rapid flow cell (11pM) and sequenced using a single read mode (SR50). This sequencing configuration was set to reach an average of 25 million reads (50-base long) per sample.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
A1023C19
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| Data processing |
Raw sequencing reads were first aligned with the bowtie2 software (v2.2.9) on the Drosophila Melanogaster (dmel-R6,21) reference genome to extract reads coming from spike-in chromatin. Remaining reads were then aligned on the Human hg38 reference genome using bowtie2 very-sensitive mode. PCR duplicates were removed from aligned data using Picard tools, and genome-browser tracks files were generated using the Deeptools suite (v3.1.0) and the bamCoverage function. In order to validate the overall H3K9me2 profile, large genomic domains were called with the EPIC software (v0.2.9, https://github.com/biocore-ntnu/epic, --window-size 500 --gaps-allowed 5 --false-discovery-rate-cutoff 0.01), and LOCKs were defined as previously proposed (McDonald et al. 2017).
Raw sequencing reads were first aligned with the bowtie2 software (v2.2.9) on the Drosophila Melanogaster (dmel-R6,21) reference genome to extract reads coming from spike-in chromatin. Remaining reads were then aligned on the Human hg38 reference genome using bowtie2 very-sensitive mode. PCR duplicates were removed from aligned data using Picard tools, and genome-browser tracks files were generated using the Deeptools suite (v3.1.0) and the bamCoverage function. Input samples per condition have been merged in a single coverage WIG file.
In order to validate the overall H3K9me2 profile, large genomic domains were called with the EPIC software [3] (v0.2.9, https://github.com/biocore-ntnu/epic, --window-size 500 --gaps-allowed 5 --false-discovery-rate-cutoff 0.01), and LOCKs were defined as previously proposed (McDonald et al. 2017).
Counts of aligned reads per human gene have been generated using the FeatureCounts software [4] and the gene coordinates extracted from the GENCODE annotation file (v26). Genes with less than 10 reads were discarded from the analysis. The raw count table was then normalized using the TMM method from the edgeR R package [5] (v3,22,3), and the limma voom (v3.36.3) functions were applied to detect genes with differential binding between untreated and EGF samples. Finally, genes with an adjusted pvalue < 0,05 and a log2 fold-change > 0,3 were called significant.
Genome_build: hg38
Supplementary_files_format_and_content: wig file of ChIP signal
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| Submission date |
Oct 23, 2018 |
| Last update date |
Oct 01, 2019 |
| Contact name |
Nicolas Servant |
| E-mail(s) |
[email protected]
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| Organization name |
Institut Curie
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| Street address |
26 rue d'ulm
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| City |
Paris Cedex 05 |
| ZIP/Postal code |
75248 |
| Country |
France |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE121662 |
Role of iron in the regulation of the epithelial-to-mesenchymal transition (ChIP-seq) |
| GSE121664 |
Role of iron in the regulation of the epithelial-to-mesenchymal transition |
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| Relations |
| BioSample |
SAMN10280098 |
| SRA |
SRX4922862 |
| Supplementary data files not provided |
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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