EXPERIMENT_TITLE: GSM5534294: RPMnuc-GM-EDTA-R2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Hi-C experiments were performed on 10-20 million cells for each condition.  Crosslinking was performed with 1% formaldehyde pre-mixed with the corresponding appropriate buffer (1x HBSS, or 1 mM EDTA + 10 mM HEPES) immediately before addition to the plates of nuclei.  After 10 minutes, crosslinking was quenched with glycine, and plates of crosslinked nuclei were washed in the same buffer corresponding to their expansion or contraction condition.  It was not possible to harvest and collect nuclei by scraping and centrifugation due to the fragility of the nuclei, particularly in the expanded case, so nuclei were lysed directly in plates by aspirating all liquid and then adding 1.8 mL of 0.3% SDS in 1x NEB2 buffer (New England Biolabs) per 10 cm dish. Dishes were incubated with gentle agitation at 37oC for 30 min.  Crosslinked material was then removed the plate by scraping. If more than one plate was to be combined to obtain enough cells, the SDS-cell mixture scraped from the first plate was transferred to the second plate for another round of incubation, so that cells from all plates were combined into a final volume of less than 3 mL.  SDS was sequestered by addition of 1.8% Triton (final concentration).  Crosslinked complexes were then divided into aliquots of 560 uL each to match other Hi-C protocols, and were then subjected to digestion with HindIII overnight (500 U per 560 uL aliquot), biotin fill-in, and dilute ligation as previously described (Belton et al. 2012). After streptavidin enrichment of biotinylated ligation junctions,  Illumina paired-end adapters were ligated onto the DNA ends and then the fragments were PCR amplified.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Low-salt swelling of isolated chromatin fibers or nuclei has been used for decades to investigate the structural properties of chromatin.  But, visible changes in chromatin appearance have not been linked to known building blocks of genome structure or features along the genome sequence.  We combine low-salt swelling of isolated nuclei with genome-wide chromosome conformation capture (Hi-C) and imaging approaches to probe the effects of chromatin extension genome-wide. Photoconverted patterns on nuclei during expansion and contraction indicate that global genome structure is preserved after dramatic nuclear volume swelling, suggesting a highly elastic chromosome topology. Hi-C experiments before, during, and after nuclear swelling show changes in average contact probabilities at short length scales, reflecting the extension of the local chromatin fiber. But, surprisingly, during this large increase in nuclear volume, there is a striking maintenance of loops, TADs, active and inactive compartments, and chromosome territories.  Subtle differences after expansion are observed, suggesting that the local chromatin state, protein interactions, and location in the nucleus can affect how strongly a given structure is maintained under stress.  From these observations, we propose that genome topology is robust to extension of the chromatin fiber and isotropic shape change, and that this elasticity may be beneficial in physiological circumstances of changes in nuclear size and volume. Overall design: Hi-C experiments on control, low salt expanded, and recontracted lymphoblast nuclei
SAMPLE_TITLE: GM-EDTA-R2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "GM12878 cells"}, {"TAG": "condition", "VALUE": "Isolated nuclei expanded by incubation in 10 mM HEPES pH 7.4 + 1 mM EDTA for 1 hour, 4 degrees C"}, {"TAG": "media", "VALUE": "RPMI, 15 percentFBS, 100 micrograms/mL penicillin-streptomycin"}, {"TAG": "cell line", "VALUE": "GM12878"}]
Pool_Member_@attributes_sample_title: GM-EDTA-R2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: GM-EDTA-R2
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