EXPERIMENT_TITLE: GSM2595586: MSC_D_HiC (proliferating); Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Hi-C was performed as previously described (Belton et al., 2012)  under conditions where nuclei remained largely intact. In brief, 25-35 million crosslinked cells (1%, 10min, RT) were lysed (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.4% NP40, 1x protease inhibitor cocktail) twice for 20 min on ice, washed with 1x NEBuffer 2 twice, and distributed into 4-5 tubes. Following SDS treatment (0.1%) for 10 min at 65°C and quenching with Triton-X for 15 min at 37°C, chromatin was digested with 600 units of HindIII overnight at 37°C under constant agitation. Next day, digestion was extended for another hour with an additional 600 units of HindIII for 1-2 hours. DNA overhangs were filled-in using a biotin-dCTP containing mix (1.5µl dATP, 1.5 µl dGTP, 1.5 µl dTTP, 37.7 µl biotin-dCTP, each 10 mM stock; Invitrogen) in the presence of 1.5 µl 50 U/µl Klenow (NEB) for 45min at 37°C, and all enzymes are inactivated by adding 86 µl 10% SDS to all tubes and incubating for 30min at 65°C. Next, each individual mixture was transferred into a 15 ml tube, incubated for 10-20min on ice to quench SDS, and a mixture containing 745 µl of 10% Triton-X, 745 µl 10x ligation buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 100 mM DTT), 80 µl of a 10 mg/ml BSA stock, 80 µl of 100 mM ATP and 5.96 ml milliQ water is added to each tube, and blunt-end ligation initiated by adding 15 µl of 5 U/µl T4 DNA ligase (Invitrogen) and incubated for 6 hours at 16°C. Crosslinks were then reversed and proteins degraded by adding 35 µl of 15mg/ml proteinase K and incubated overnight at 65°C. Next day, an additional 35 µl of proteinase K was added for 2 hours at 65°C, mixtures were cooled to room temperature, transferred to 50 ml conical tubes, and DNA was extracted twice with phenol:chloroform (ph 8.0) and ethanol precipitated. The resulting pellets were dissolved in 450 µl 1x TE, transferred to 1.7ml tubes, DNA was re-extracted twice, precipitated, pellets were washed once in 70% ethanol, air-dried, and resuspended in 25 µl 1x TE. Subsequently, three tubes, each with ~5 µg of the Hi-C template, were incubated for 2 h at 12°C in the presence of 1 µl 10 mg/ml BSA, 10 µl 10x NEBuffer 2, 1 µl 10 mM dATP, 1 µl 10 mM dGTP (for NcoI samples dCTP was used instead) and 5 units of T4 DNA polymerase (NEB) in a total volume of 100 µl to remove biotin-16-dCTP at non-ligated DNA ends. Reactions were stopped by adding 2 µl of 0.5 M EDTA pH 8.0, and the DNA was subsequently purified by phenol:chloroform extraction followed by ethanol precipitation. DNA pellets were resuspended and pooled in a total of 100µl of milliQ water, and residual salts were removed via Amicon 30K columns using three washing steps (1x 200 µl and 2x 100 µl milliQ water). Finally, DNA was sheared to a size of 300-600 bp on a Bioruptor Plus (Diagenode; 2x 10 cycles of 30 sec on and 30 sec off, at the highest power setting). For IMR90 cells, the procedure was slightly modified to follow the in situ Hi-C protocol (Rao et al. 2014). Approx. 20 million cells were lysed twice in 900 µl lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.4% NP-40, 1x protease inhibitor cocktail) for 15 min on ice, nuclei were pelleted and washed once in 1x NEBuffer 2, distributed into 4 tubes, resuspended in 200 µl 0.5% SDS/NEBuffer 2, and incubated for 10 min at 62°C. After repelleting, SDS-treated nuclei were resuspended in 150µl 1% Triton-X/NEBuffer 2, and digested overnight using 400 units of HindIII at 37°C with constant agitation. Next day, another 400 units of HindIII were added for another 1-2 h at 37°C. Following heat inactivation for 20 min at 62°C, nuclei were pelleted by centrifugation and resuspend in 256 µl NEBuffer 2. DNA ends were then biotinylated by adding 50 µl of a biotin-dCTP-Klenow mix (37.5µl of 0.4 mM biotin-dCTP, 1.5µl of 10 mM dTTP, dATP, and dGTP, and 1.5µl of 50 U/μl Klenow) to each tube and incubating for 1 h at 37°C. Subsequently, 900 µl of ligation mix (603 µl H2O, 240 µl 5x Invitrogen ligation buffer, 30 µl of 10% Triton-X, 12 µl of 10 mg/ml BSA, and 15 µl ligase) was added to each sample, mixed, and incubated for 6 h at 16°C; tubes were inverted frequently. Nuclei were next pelleted and a volume of 900 µl was removed. Following reversal of crosslinking and proteinase K addition, DNA is phenol:chloroform extracted and ethanol precipitated; all subsequent steps were performed as described above. Hi-C DNA was used as template for adding sequencing linkers via only 10 PCR cycles. Then, Hi-C libraries were sequenced to generate at least 400 million paired-end reads (50-75 bp in length) per each donor/condition in a HiSeq4000 platform (Illumina).
STUDY_DESCRIPTOR_STUDY_ABSTRACT: We hypothesized that entry into senescence of different primary human cells can be triggered by one early molecular event affecting the spatial organization of chromosomes. To test this, we combined whole-genome chromosome conformation capture, population and single-cell transcriptomics, super-resolution imaging, and functional analyses applied on proliferating and replicatively-senescent populations from three distinct human cell types. We found a number of genes involved in DNA conformation maintenance being suppressed upon senescence across cell types. Of these, the abundant high mobility group (HMG) B1 and B2 nuclear factors are quantitatively removed from cell nuclei before typical senescence markers appear, and mark a subset of topologically-associating domain (TAD) boundaries. Their loss coincides with obvious reorganization of chromatin interactions via the dramatic spatial clustering of CTCF foci. HMGB2 knock-down recapitulates this senescence-induced CTCF clustering, while also affecting insulation at TAD boundaries. Overall design: Hi-C was generated in HUVEC (D1-D3), IMR90 (I10, I79) and MSC (D) using HindIII restriction digestion and HMGB2 knockdown HUVEC (NTC, siHMGB2)
SAMPLE_TITLE: MSC_D_HiC (proliferating)
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "mesenchymal stromal cells"}, {"TAG": "cell type", "VALUE": "mesenchymal stromal cells"}, {"TAG": "donor", "VALUE": "single donor"}, {"TAG": "treatment", "VALUE": "proliferating"}, {"TAG": "genotype", "VALUE": "WT"}]
Pool_Member_@attributes_sample_title: MSC_D_HiC (proliferating)
RUN_SET_RUN_Pool_Member_@attributes_sample_title: MSC_D_HiC (proliferating)
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