EXPERIMENT_TITLE: GSM4140493: E2_60_HiC_run2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Adherent cells were directly cross-linked on the plates with 1% formaldehyde for 10min at room temperature. After addition of glycine (125mM final) to stop the reaction, cells were washed with PBS and recovered by scrapping. Cross-linked cells were incubated 30min on ice in 3C lysis Buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% NP40, 1X anti-protease cocktail), centrifuged 5min at 3,000 rpm and resuspended in 190uL of NEBuffer2 1X  (New England Biolabs – NEB). 10 µL of 10% SDS were added and cells were incubated for 10min at 65ºC. After addition of Triton X-100 and 15min incubation at 37ºC, nuclei were centrifuged 5min at 3,000 rpm and resuspended in 300uL of NEBuffer2 1X. Digestion was performed overnight using 400 U MboI restriction enzyme (NEB). To fill-in the generated ends with biotinylated-dATP, nuclei were pelleted and resuspended in fresh repair buffer 1x (1.5µL of 10mM dCTP; 1.5µL of 10mM dGTP; 1.5µL of 10mM dTTP; 37.5µL of 0.4mM Biotin-dATP; 50U of DNA Polymerase I Large (Klenow) fragment in 300uL NEBuffer2 1X). After 45min incubation at 37ºC, nuclei were centrifuged 5min at 3,000 rpm and ligation was performed 4h at 16ºC using 10,000 cohesive end units of T4 DNA ligase (NEB) in 1.2mL of ligation buffer (120µL of 10X T4 DNA Ligase Buffer; 100µL of 10% Triton X-100; 12µL of 10mg/mL BSA; 963µL of H2O). After reversion of the cross-link, DNA was purified by phenol extraction and EtOH precipitation. Purified DNA was sonicated to obtain fragments of an average size of 300-400 bp using a Bioruptor Pico (Diagenode; 8 cycles; 20s on and 60s off). 3µg of sonicated DNA was used for library preparation. Biotinylated DNA was pulled down using 20uL of Dynabeads Myone T1 streptavidine beads in Binding Buffer (5mM Tris-HCl pH7.5; 0.5mM EDTA; 1M NaCl). End-repair and A-tailing were performed on beads using NEBnext library preparation end-repair and A-tailing modules (NEB). Illumina adaptors were ligated and libraries were amplified by 8 cycles of PCR. Resulting Hi-C libraries were first controlled for quality by low sequencing depth on a NextSeq500 prior to higher sequencing depth on HiSeq2000.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Estrogen (E2) and Progesterone (Pg) via their specific receptors, ER and PR respectively, are major determinants in the development and progression of endometrial malignancies. We have studied how E2 and the synthetic progestin R5020 affect genomic function in Ishikawa endometrial cancer cells. Using ChIPseq in cells exposed to the corresponding hormones, we identified cell specific binding sites for ER (ERbs) and PR (PRbs), mostly binding to independent sites and both adjacent to PAXbs. Long-range interactions (HiC) showed enrichment of PRbs and PAXbs, which we call progestin control regions (PgCRs) inside TADs with differentially progestin-regulated genes. Effects of hormone treatments on gene expression were detected by RNAseq. PgCRs correlate with open chromatin independently of hormonal stimuli. In summary, endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR to compartmentalized PgCRs in hormone-independent open chromatin, which include binding of partner transcription factors, in particular PAX2. Overall design: Endometrial adenocarcinoma Ishikawa cells were treated with R5020 10nM or E2 10nM for different times and submitted to Hi-C.
SAMPLE_TITLE: E2_60_HiC_run2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "endometrial adenocarcinoma"}, {"TAG": "disease state", "VALUE": "endometrial adenocarcinoma"}, {"TAG": "cell line", "VALUE": "Ishikawa"}, {"TAG": "passages", "VALUE": "20-26"}, {"TAG": "cell type", "VALUE": "epithelial-like"}, {"TAG": "growth mode", "VALUE": "adherent"}, {"TAG": "treatment", "VALUE": "60min E2-treated Ishikawa cells"}]
Pool_Member_@attributes_sample_title: E2_60_HiC_run2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: E2_60_HiC_run2
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