EXPERIMENT_TITLE: GSM3436597: MCF7_SatE2_R1: 3e Hi-C human Saturated E2 MCF7 cell rep.1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: fresh MCF7 cells were cross-linked with 1% formaldehyde for 10 minutes at 25°C.  2-5 X 106 cells were lysed in 10 ml lysis buffer (10 mM Tris-HCl pH8.0, 10 mM NaCl, 0.2% NP40; 10 μl protease inhibitors (Sigma)) with rotation at 4°C for 60 minutes. The cells were then treated with 400 μl 1X NEB cutsmart buffer with 0.1% SDS at 65°C for 10 minutes, followed by addition of 44 μl 10% Triton X-100 to quench SDS. Chromatin was subsequently digested with 20 Units CviQ I (NEB), and 20 Units CviA II (NEB) at 25°C for 20 minutes, then with 20 Units Bfa I (NEB) at 37°C for 20 minutes. The reaction was stopped by washing the cells twice with 600 l wash buffer (10mM NaCl, 1mM EDTA, 0.1% triton-100). The DNA ends were blunted and labeled with biotin by Klenow enzyme in the presence of dCTP, dGTP, dTTP, biotin-14-dATP, followed by ligation using T4 DNA ligase. After reverse crosslinking, the samples were treated with T4 DNA polymerase to remove biotin labels at the DNA ends. DNA was fragmented to 300-500 bp by sonication with a Bioruptor sonicator (Diagenode UCD-200). Next, The DNA was end-repaired, followed by A-addition as described previously (Lieberman-Aiden et al., 2009). The remaining biotinylated DNA fragments were then captured using Dynabeads MyOne Streptavin C1 Beads (Invitrogen) by incubating for 30 minutes at 25°C with rotation. The DNA on beads was ligated to the Illumina Paired End Adaptors. Following PCR-amplification of the libraries, DNA fragments from 300 to 700 bp were purified from 2% E-gel and sequenced on Hi-Seq 3000.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Transcriptional regulation in metazoans occurs through long range genomic contacts between enhancers and promoters, and most genes are transcribed in episodic 'bursts' of RNA synthesis. The relationship between these two phenomena and the dynamic regulation of genes in response to upstream signals is unknown. Here, we describe the use of live-cell RNA imaging coupled with Hi-C measurements to dissect the regulation of the estrogen-responsive TFF1 gene under endogenous regulation. Although this gene is highly induced, we observe short active periods and variable inactive periods ranging from minutes to days. The heterogeneity in inactive times gives rise to the widely-observed 'noise' in human gene expression and explains the distribution of protein levels in human tissue. We derive a mathematical model of regulation that relates transcription, chromosome structure, and the cell's ability to 'sense' changes in estrogen and predicts that hypervariability is largely dynamic and does not reflect a stable biological state. Overall design: We used 3e HiC to look at the estradiol induced entropy changes for ER bound enhancers.
SAMPLE_TITLE: MCF7_SatE2_R1: 3e Hi-C human Saturated E2 MCF7 cell rep.1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "MCF7 breast cancer cells"}, {"TAG": "cell line", "VALUE": "MCF7"}, {"TAG": "tissue", "VALUE": "breast cancer"}, {"TAG": "gender", "VALUE": "female"}]
Pool_Member_@attributes_sample_title: MCF7_SatE2_R1: 3e Hi-C human Saturated E2 MCF7 cell rep.1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: MCF7_SatE2_R1: 3e Hi-C human Saturated E2 MCF7 cell rep.1
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