EXPERIMENT_TITLE: GSM7635705: CT29, EVT, rep3; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Cells were rinsed with 5 mL of PBS and then 5 mL PBS containing 2% formaldehyde were added and incubated for 10 min. Then, 550 μL of glycine 1.25 M (final conc 0.125M) were added and incubated for 5 min at room temperature before an additional 15 min incubation on ice. Liquid was removed and cells were rinsed two times with 5 mL ice-cold PBS. Cells were scraped and transferred into 2 mL low bind tubes to have the equivalent of 1*10^6 cells (1 Petri Dish). Nuclei were pelleted at 2,500 rpm for 10 min at 4˚C (repeat if necessary) and all supernatant was removed, and flash frozen on dry ice and stored at -80˚C until ready for use. Crosslinked samples were thawed on ice and prepared for Hi-C using the Arima-HiC kit (Arima Genomics, San Diego, CA). Cross-linking was performed according to the Arima-HiC protocol with the following modifications. At step 5, the incubation at 37°C was increased to a duration of 60 min and the samples were maintained overnight at 4°C after step 11. Samples were then cleaned with AMPure XP Beads as described in the protocol and all quantification and quality control steps that required the use of a Qubit were performed using a Qubit dsDNA HS Assay Kit. Proximally ligated DNA (1000 ng/sample) was used as an input into the library preparation protocol. Samples were sheared to 400 bp using a Covaris LE220-plus system using the manufacturer's recommended settings. Sizing was confirmed using a High Sensitivity DNA Bioanalyzer kit. Size-selection as described in the Arima-HiC protocol was followed and the entire size-selected sample was used as input into biotin enrichment and library preparation with a KAPA HyperPrep kit and Roche SeqCap Adapters (7141530001 or 7141548001, Roche). Instead of performing the Arima-HiC quality control step 2, the number of cycles needed for library amplification was determined as follows: perform 10 PCR cycles for 125-200 ng of input, perform 9 PCR cycles for 200-400 ng of input, perform 8 PCR cycles for 400-600 ng of input, and perform 7 cycles of PCR for inputs greater than 600 ng. The completed libraries were then quantified using a Qubit dsDNA BR Assay Kit and the library size was determined with a High Sensitivity DNA Bioanalyzer Kit. Standard Illumina Free-Adapter Blocking was performed on the libraries. Cleaned, adapter-blocked libraries were loaded on a NovaSeq6000 with a run configuration of 151x8x8x151 and an average depth of 900M PE reads per library.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The extravillous trophoblast (EVT) cell lineage is a key feature of placentation and critical for spiralartery remodeling and successful pregnancy. Our knowledge of transcriptional regulation driving EVT cell development is limited. Here, we mapped the transcriptome and epigenome landscape as well as chromatin interactions of human trophoblast stem (TS) cells and their transition into the differentiated EVT cell lineage. We identified that chromatin accessibility in intergenic regions was more extensive in EVT cells than in TS cells in the stem state, which is indicative of increased enhancer-driven gene regulation. Using reference epigenome annotation, we noted that 18% of the chromatin landscape in EVT cells was uncharted. We linked regulatory regions to their cognate target genes and characterized the three-dimensional organization of the TS cell functional genome by Hi-C. Integrational analysis of chromatin accessibility, long-range chromatin interactions, transcriptomic, and transcription factor binding motif enrichment enabled identification of transcription factors and regulatory mechanisms associated with EVT cell lineage development. Subsequent analyses elucidated functional roles forTFAP2C,EPAS1,SNAI1, andDLX6in the regulation of EVT cell lineage development.EPAS1was identified as an upstream regulator of EVT cell transcription factors, includingSNAI1andDLX6, and was found to be upregulated in idiopathic recurrent pregnancy loss. Collectively, we have revealed activation of a dynamic regulatory network that provides a framework for understanding EVT cell specification in trophoblast cell lineage development and human placentation. Overall design: Hi-C on stem state and extravillous trophoblast cells from cell line CT27 and CT29
SAMPLE_TITLE: CT29, EVT, rep3
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "human trophoblast stem cells"}, {"TAG": "tissue", "VALUE": "human trophoblast stem cells"}, {"TAG": "cell line", "VALUE": "CT29"}, {"TAG": "cell type", "VALUE": "extravillous trophoblast cell"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: CT29, EVT, rep3
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