EXPERIMENT_TITLE: GSM2645715: Hi-C MCF-7 +E2 replicate 2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Hi-C, samples were fixed for 10 minutes with 1% formaldehyde. Fixation was stopped by adding Glycine (0.125M). Samples were frozen at -80 degee Celcius till used. In situ Hi-C was essentially performed as described(Rao et al., 2014). Briefly, for each experiment, 2 x 10E6 cells, fixed for 10 minutes with 1 % formaldehyde/PBS and washed twice with PBS, permeabilized for 7 minutes at 62°C in a PCR cycler with 200 µl lysis buffer (0.5% SDS, 50 mM Tris-HCl pH=7.5, 10 mM NaCl, 1mM EDTA, and 1X protease inhibitors solution (Roche)) and pelleted at 2500x g for 5 minutes. Supernatant was removed and nuclei were resuspended in 25 µl 10% Triton X-100, 25 µl NEB 2 buffer, 195 µl water, and rotated for 15’ at 37°C. Chromatin was digested overnight at 37°C after adding 0.5 µl 1 M DTT and 4 µl 25 U/µl Mbo I and rotated at 8 RPM. MboI was inactivated by incubation at 62°C for 20 minutes. Nuclei were centrifuged for 5 minutes at 500 x g and 200 µl supernatant was discarded. Overhangs were filled in by adding 32 μL water, 5 μl of 10X NEBuffer2, 0.35 μl of 10 mM dATP, 0.35 μl of 10 mM dTTP, 0.35 μl of 10 mM dGTP, 7.5 μl 0.4 mM Biotin-14-dCTP (Invitrogen), 4 μl 10% Triton X-100, and 5 μl of 5 U/μl Klenow enzyme (Enzymatics) and rotating for 40 minutes at room temperature. The reaction was stopped by adding 2.5 µl 0.5 M EDTA. DNA was ligated under rotation overnight at 16°C in a total volume of 400 µl ligase mix  containing 40ul 10x T4 DNA ligase buffer (Enzymatics), 36 µl 10 % Triton X-100, 5 µl 100x BSA (10 mg/ml), 1 µl (1200 U) T4 DNA ligase (Enzymatics). The reaction was terminated by adding 20 µl 0.5 M EDTA. Samples were digested for 15 minutes at 42°C with 1 µl 10 µg/µl RNase A. 33 μl of 5 M sodium chloride and 55 µl of 10% SDS were added and reverse crosslinked for 4 h at 65°C. Protein was digested with 10 μl of 20 mg/ml proteinase K (Life Technologies), incubated at 55°C for 120 minutes, shaking at 800 RPM, then 65°C for 90 minutes. DNA was extracted once with 600 µl phenol/chloroform/isoamylalcohol (25:24:1) Tris-buffered to pH 8.0 and once with 300 µl CHCl3, and precipitated overnight at -20°C with 1.5 µl 20 mg/ml glycogen and 1412 µl 100% ethanol overnight. DNA was pelleted for 20 minutes at 16000x g, 4°C and washed once with 1 ml 80% ethanol for 5 minutes, 8000x g, 4°C. Pellets were dissolved in 131 µl TT (0.05% Tween 20/10 mM Tris pH=8) each. DNA was sheared with 300 bp Covaris protocol in snap cap tube in a Covaris E220 at 10 % duty cycle, intensity 140 W, 200 cycles/burst for 80” total time. Large DNA fragments (>400 bp) were depleted with 5 µl Speedbeads and 6.45% PEG8000/2.5 M NaCl. Supernatant was transferred to fresh tubes and small DNA fragments were collected with 9.5% PEG8000 by adding an additional 60 µl PEG8000/2.5 M NaCl and 3 µl Speedbeads. DNA was eluted in 50ul TT for 5 minutes. DNA was captured with 50 µl 2x B&W buffer containing 0.2 % Tween 20 and 15 µl T1 Dynabeads (Invitrogen, washed twice with 1x B&W buffer (10 mM Tris-HCl pH 7.5, 01 mM EDTA, 2 M NaCl, then suspended in 51 µl 2x B&W buffer containing 0.2% Tween 20), rotating for 30 minutes at room temperature. Beads were washed once with 500 µl each of 1x B&W/0.1 % Triton-X100, once with TET (0.05% Tween 20/TE). Hi-C library bound to beads were resuspend in 100 µl end repair mix (KAPA Library Preparation for Illumina) Incubated for 30 minutes at 20°C. Reaction was stopped by adding 2.5 µl 0.5 M EDTA. Beads were collected and washed twice with 150 µl 1x B&W/0.1% Triton-X100, once with 180 µl TET. Beads were resuspend in 50 ul A-tailing reaction mix (KAPA Library Preparation for Illumina), incubated 30 minutes at 30°C. Reaction was stopped by adding 1.5 µl 0.5 M EDTA. Beads were collected and washed twice with 150 µl 1x B&W/0.1% Triton-X100, once with 180 µl TET. Sequencing adapters were ligated to the bead-bound DNA in 100 µl 1x rapid ligation buffer (Enzymatics) containing 0.1% Tween 20,  2ul 1:20 Truseq adapters (Illumina), 1 µl (3000 U) T4 DNA ligase (Enzymatics) for 20 minutes at room temperature. The reaction was stopped with 5 µl 0.5 M EDTA, beads washed twice with 1x B&W, twice with 0.1% Tween 20/TE, then resuspended in 30 µl 0.033% Tween20/LoTE, (TE diluted 1:4 with water). Libraries were PCR-amplified using the 10 µl of the bead suspension as template for 10 cycles using KAPA HiFi, size-selected to 225-425 bp insert size using speedbeads PEG8000/2.5 M NaCl solutions, and paired-end sequenced on an Illumina HiSeq 2500.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Detailed analysis of estrogen mediated enhancer landscape and chromosomal architectural changes specifically in human chromosome 21. Overall design: in situ Hi-C experiments are designed to elucidate 3D chromatin landscape in human breast cancer cells (MCF7) under basal and estrogen treated conditions.
SAMPLE_TITLE: Hi-C MCF-7 +E2 replicate 2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Hi-C MCF-7 +E2"}, {"TAG": "cell line", "VALUE": "MCF7"}, {"TAG": "cell type", "VALUE": "Human breast cancer cells"}, {"TAG": "genotype/variation", "VALUE": "wild type"}, {"TAG": "treated with", "VALUE": "estrogen; Treatement"}]
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