EXPERIMENT_TITLE: GSM7900697: Hi-C for cell grown in 3D normal media; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Hi-C was performed as previously described (11) with the following modifications: (i) two million cells obtained from T47D cells cultured in 2D monolayer or as 3D spheroids were used as starting material; (ii) chromatin was initially digested with 100 U MboI (New England BioLabs) for 2 h, and then another 100 U (2 h incubation) and a final 100 U were added before overnight incubation; (iii) before fill-in with bio-dATP, nuclei were pelleted and resuspended in fresh 1 NEB2 buffer; (iv) ligation was performed overnight at 24°C with 10,000 cohesive end units per reaction; (v) de-cross-linked and purified DNA was sonicated to an average size of 300–400 bp with a Bioruptor Pico (Diagenode; seven cycles of 20 sec on and 60 sec off); (vi) DNA fragment-size selection was performed only after final library amplification library preparation was performed with an NEBNext DNA Library Prep Kit (New England BioLabs) with 3 μl NEBNext adaptor in the ligation step; (viii) libraries were amplified for 8–12 cycles with Herculase II Fusion DNA Polymerase (Agilent) and were purified/size-selected with Agencourt AMPure XP beads (>200 bp). Hi-C library quality was assessed through ClaI digestion and lowcoverage sequencing on an Illumina NextSeq500 instrument, after which every technical replicate (n=2) of each biological replicate (n=2) was sequenced at high coverage on an Illumina HiSeq2500 instrument. Data from technical replicates were pooled for downstream analysis. We sequenced >18 billion reads in total to obtain 0.78–1.21 billion valid interactions per time point per biological replicate.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The cancer epigenome has been studied in cells cultured in 2D monolayers on plastic surfaces, but recent studies highlight the impact of the extracellular matrix (ECM) and the 3D environment on multiple cellular functions. Here, we report the physical, biochemical and genomic differences between T47D breast cancer cells cultured in 2D monolayer and as 3D spheroids in Matrigel. Cells within 3D spheroids exhibit a rounder nucleus with less accessible, more compacted chromatin, and altered expression of over 2,000 genes, the majority of which become repressed. Hi-C analysis reveals that cells grown in 3D exhibit enrichment in regions belonging to the B compartment, decrease on chromatin bound CTCF and increased fusion of Topologically Associating Domains (TADs). Upregulation of the Hippo pathway in 3D spheroids results in the activation of the LATS1 kinase, which promotes phosphorylation and displacement of CTCF from DNA, likely responsible for the observed TAD fusions. Cells grown in 3D exhibit higher progesterone receptor (PR) binding to chromatin, leading to an increase in the number hormone-regulated genes. This effect is in part mediated by the LATS1 activation, which favors cytoplasmic retention of YAP and CTCF removal. Overall design: Epigenomic analysis of LATS1 kinase in controlling CTCF for 3D growth of breast cancer cells
SAMPLE_TITLE: Hi-C for cell grown in 3D normal media
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "breast"}, {"TAG": "tissue", "VALUE": "breast"}, {"TAG": "cell line", "VALUE": "T47D"}, {"TAG": "cell type", "VALUE": "Epithelial cancer metastatic"}, {"TAG": "treatment", "VALUE": "3D culture normal media"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: Hi-C for cell grown in 3D normal media
RUN_SET_RUN_Pool_Member_@attributes_sample_title: Hi-C for cell grown in 3D normal media
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