EXPERIMENT_TITLE: GSM2973930: Neu-D1_HiC_rep1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: For RNA-seq, total mRNA was isolated from Neu-D1 and Neu-D3 cells using the Qiagen RNeasy kit and processed for Illumina library preparation and sequencing. For ChIP-seq of lamin B1, cells at each differentiation time point were fixed with 1% formaldehyde for 10 min, lysed for 10 min in ChIP lysis buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS, protease inhibitors and 1 mM PMSF) and sonicated 4 times 10 min in a Bioruptor (Diagenode) to generate 200 base-pair DNA fragments. After sedimentation, the supernatant was collected and diluted 10-fold in RIPA (140 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, protease inhibitors, 1 mM PMSF). Chromatin was incubated overnight at 4oC with the anti-lamin B1 antibody coupled to Dynabeads Protein A/G (Invitrogen). ChIP samples were washed 4 times in ice-cold RIPA, crosslinks reversed and DNA eluted for 6 h at 68oC in 50 mM NaCl, 20 mM Tris-HCl pH 7.5, 5 mM EDTA, 1% SDS and 50 ng/µl Proteinase K. DNA was purified using phenol-chloroform isoamylalcohol and dissolved in H2O prior to processing for Illumina library preparation and sequencing. For Hi-C, 25 million cells per time point and differentiation replicate were cross-linked with fresh 1% formaldehyde (Pierce) for 12 min at room temperature on a rocker. Excess formaldehyde was quenched with 0.125 M glycine for 5 min and cells washed 3 times 5 min in ice-cold PBS. Crosslinked cells were lysed in lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630 and protease inhibitors) on ice for 15 min and homogenised by Douncing with a B-pestle (tight) in lysis buffer until nuclei were released, as assessed by microscopy. The lysate was sedimented, the pellet was resuspended in NEBuffer 2 (10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol) containing 0.1% SDS, and incubated at 65oC for 10 min. SDS was quenched on ice with 1% Triton X-100 and the sample digested with HindIII overnight at 37oC. Digested fragments were labelled with biotin-14-dCTP and blunt-end ligated. Crosslinks were reversed at 65oC overnight, the ligated DNA was purified by phenol-chloroform:isoamylalcohol (Ambion), desalted and RNAse-treated. DNA was sheared to ~100-300 base-pairs by Covaris sonication and size-fractionated on AMpure XP magnetic beads (Beckman Coulter). Biotinylated DNA fragments were pulled-down with Streptavidin beads and processed for Illumina adapter ligation and library preparation. Illumina sequencing libraries were generated as per Illumina protocols.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: How genomic information is selectively utilized to direct spatial and temporal gene expression patterns during differentiation remains to be elucidated but it is clear that regulated changes in higher-order genomic architecture plays a fundamental role. Specifically, long range interactions within and between chromosomes and the position of chromosome territories in the nucleus are controlled by TADs and LADs respectively, but the relationship between these genomic organizers remains poorly understood Overall design: We analyzed the large-scale spatial reorganization of chromatin by generating matched Hi-C and nuclear lamin-chromatin contact datasets throughout a dual adipose/neuronal induction of human primary adipose stem cells.  We have mapped Hi-C (TADs) and lamin-associated domains (LADs) in multiple steps during adipose stem cell differentiation to characterize the spatial and temporal link between genomic architecture and gene expression. We identify a new level of 4D genomic organization involving a long-range clustering of individual TADs or TAD pairs into TAD cliques. LADs appear to regulate their formation. (ASCs). We unveil a lineage-specific dynamic assembly and disassembly of repressive cliques of linearly non-contiguous TADs, and a time course-coupled relationship between TAD clique size and lamina association. Our findings reveal a new level of developmental genome organization and provide an overview of large-scale changes in the 4D nucleome during lineage-specific differentiation.
SAMPLE_TITLE: Neu-D1_HiC_rep1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Human primary adipose stem cells"}, {"TAG": "time point", "VALUE": "Day 1"}, {"TAG": "cell type", "VALUE": "ASCs on day 1 after neuronal induction"}, {"TAG": "chip antibody", "VALUE": "N/A"}, {"TAG": "replicate", "VALUE": "rep1"}]
Pool_Member_@attributes_sample_title: Neu-D1_HiC_rep1
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