EXPERIMENT_TITLE: GSM5518583: DKO_HiC_rep3; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: HiC protocol was performed similar to (Rao et al., 2014). In brief, 4M fixed cells were then resuspended in 500ul lysis buffer (50mM Tris-HCl pH 8.0, 10mM NaCl, 0.2% NP40, 1X PITC) for 30 min on ice with periodic resuspension. Lysed cells were spun 3500 RPM for 3 min and resuspended in 300 μl 1× NEB buffer 2, spun and resuspended in 198 μl 1× NEB buffer 2. 2μl of 10% SDS was added and incubated at 65°C for 10 min. After, 400 μl 1× NEB buffer 2 and 60 μl 10% Triton X-100 were added to quench the SDS. Samples were incubated at 37°C for 15 min. Nuclei were spun 3500 RPM for 3 min and resuspended in 300 μl 1× NEB buffer 2, repeat wash step. Fixed nuclei were then digested with MboI (NEB#R0147M) overnight, 5' overhangs were filled-in with a biotinylated nucleotide, blunt-ends were ligated, followed by reverse crosslinking overnight. The purified DNA (2 μg) was sonicated using Covaris 50μl 400bp protocol. The sonicated DNA was brought to a volume of 400μl in binding buffer (5 mM Tris-HCl pH7.5; 0.5 mM EDTA; 1M NaCl) and mixed with 20μl of streptavidin magnetic beads (NEB#S1421) and rotated for 1hr at RT. The bead-bound DNA was washed twice with 400μl low-TE and resuspended in 50μl low-TE. NGS libraries were prepared using NEB DNA Ultra II kit (NEB#E7645). End prep: Mix 50μl of sample with 7μl End prep buffer and 3μl End prep enzyme, incubate for 30 min at RT then 30 min at 65°C, wash twice with 400μl low-TE and resuspended in 60μl low-TE. Adaptor ligation: 2.5μl of adaptor and 30μl of ligation mix was incubated at RT for 1-3hrs, washed twice with low-TE and resuspended in 90μl low-TE, following ligation, 3 μl USER was added for 30mins at 37°C, wash twice with 400μl and resuspend in 15μl. PCR: Add 5μl of universal F and index R primer, 25μl Q5 mix, 15μl sample for 5 PCR cycles. Libraries were purified with SPRI beads (0.9X) and quantified on a bioanalyzer and with NEB Illumina quant kit (NEB#E7630). HiC libraries were sequenced on a NextSeq500 either 150bp or 75bp paired-end reads. Libraries were prepared using NEB UltraII DNA library kit
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Two dominant processes organizing chromosomes are loop extrusion and the compartmental segregation of active and inactive chromatin. The molecular players involved in loop extrusion during interphase, cohesin and CTCF, have been extensively studied and experimentally validated. However, neither the molecular determinants nor the functional roles of compartmentalization are well understood. Here, we distinguish three inactive chromatin states using contact frequency profiling, comprising two types of heterochromatin and a previously uncharacterized inactive state exhibiting a neutral interaction preference. We find that heterochromatin marked by long continuous stretches of H3K9me3, HP1a and HP1ß correlates with a conserved signature of strong compartmentalization and is abundant in HCT116 colon cancer cells. We demonstrate that disruption of DNA methyltransferase activity dramatically remodels genome compartmentalization as a consequence of the loss of H3K9me3 and HP1 binding. Interestingly, H3K9me3-HP1a/ß is replaced by the neutral inactive state and retains late replication timing. Furthermore, we show that H3K9me3-HP1a/ß heterochromatin is permissive to loop extrusion by cohesin but refractory to CTCF, explaining a paucity of visible loop extrusion-associated patterns in Hi-C. Accordingly, CTCF loop extrusion barriers are reactivated upon loss of H3K9me3-HP1a/ß, not as a result of canonical demethylation of the CTCF binding motif but due to an intrinsic resistance of H3K9me3-HP1a/ß heterochromatin to CTCF binding. Together, our work reveals a dynamic structural and organizational diversity of the inactive portion of the genome and establishes new connections between the regulation of chromatin state and chromosome organization, including an interplay between DNA methylation, compartmentalization and loop extrusion. Overall design: We compare genome structure and chromatin state genome-wide using Hi-C, ChIP-seq, Repli-seq, and Protect-seq in HCT116 colon cancer cells with its genetic derivative DKO cells which lack DNMT3B and DNMT1 activity and HCT116 cells treated with the DNA methylation inhibitor 5-Azacytidine (5Aza).
SAMPLE_TITLE: DKO_HiC_rep3
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "DKO"}, {"TAG": "cell type", "VALUE": "colon cancer cell line"}, {"TAG": "treatment", "VALUE": "none"}, {"TAG": "Sex", "VALUE": "male"}, {"TAG": "enzyme", "VALUE": "MboI"}]
Pool_Member_@attributes_sample_title: DKO_HiC_rep3
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