EXPERIMENT_TITLE: GSM5456349: K562_EZH2i_plus_dox_rep2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Cells were fixed in 1% formaldehyde, washed, snap frozen, and stored at -80ºC. Nuclei were lysed, and overnight digestion was performed using DpnII. Digested ends were filled in with biotinylated dATP and in situ ligation was performed. Samples were digested with Proteinase K for and crosslinks were reversed. DNA was purified through an ethanol precipitation and sheared to a size of 300-500 bp with sonication. DNA was size-selected with a double-SPRI magnetic bead purification. At this point, the DNA was split in half for bisulfite (LIMe) and traditional in situ Hi-C, if performed. For the traditional in situ Hi-C workflow, biotinylated fragments were isolated using T1 streptavidin beads, and on bead library preparation was performed on beads according to published precedent. The preparation of bisulfite (LIMe) Hi-C libraries was performed according to the Hi-Culfite protocol. DNA was bisulfite-converted with the EpiTect Fast Bisulfite Conversion Kit (Qiagen). Following bisulfite conversion, biotinylated fragments were purified using C1 streptavidin beads. LIMe-Hi-C libraries were generated using the Accel-NGS-methyl-Seq Kit (Swift Biosciences) according to the manufacturers protocol with 8-10 cycles of amplification. LIMe-Hi-C and in situ Hi-C
STUDY_DESCRIPTOR_STUDY_ABSTRACT: We developed and performed LIMe-Hi-C in order to assess lamina association, DNA methylation, and chromosome conformation in one experimental workflow. We combined LIMe-Hi-C with chemcial inhibition of EZH2 and DNMT1 to better understand the influence of H3K27me3 and DNA methylation on 3D genome organization. Overall design: LIMe-Hi-C experiments were performed in triplicate for FLAG-NLS-M.CviPI-Lamin-B1 K562 cells. A no doxycycline triplicate condition was included as a control to assess the specificity of the  lamina association signal. EZH2 inihibtion, DNMT1 inhibition and vehicle (DMSO) treatment were each performed in triplicate for 72 hours with doxycycline added for the final 24 hours of the drug treatment. The DMSO + doxyxline condition was utilized as a control. An in situ Hi-C experiment in singlicate for the DMSO + doxycyline condition was included as quality control for the bisulfite Hi-C workflow.
SAMPLE_TITLE: K562_EZH2i_plus_dox_rep2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Flag-NLS-M.CviPI-Lamin-B1 K562 clonal cells, EZH2i (GSK343 ) 72 hours, Doxycycline 24 hours"}, {"TAG": "cell line", "VALUE": "Flag-NLS-M.CviPI-Lamin-B1 K562 clonal cells"}, {"TAG": "cell type", "VALUE": "chronic myeloid leukemia"}, {"TAG": "treatment", "VALUE": "Flag-NLS-M.CviPI-Lamin-B1 K562 clonal cells were treated with GSK343 (1\u00b5M) for 72 hours and induced with doxcycline (1\u00b5g/mL) for the final 24 hours of treatment."}]
Pool_Member_@attributes_sample_title: K562_EZH2i_plus_dox_rep2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: K562_EZH2i_plus_dox_rep2
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