EXPERIMENT_TITLE: GSM8789268: donor1, IL2, 250000 cells, replicate 2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Cells were counted and spun into pellets of the required numbers (50000, 250000, or 1000000) at 5000xg for 5 mins. Supernatant was removed until <20ul of liquid + cell pellet remained. The pellets were snap frozen on dry ice and then stored in at -70oC until processing. All pellets were committed to the mammalian cell low input Arima Genomics Inc workflow (https://arimagenomics.com/wp-content/files/User-Guide-Arima-HiC-for-Mammalian-Cell-Lines.pdf, September, 2024) with the following modifications; conditioning incubation time 20 mins, restriction enzyme cutting time 1hr. Proximally ligated DNA samples were QC for biotin nucleotide incorporation if enough material was available. Sonication was done using a S220 Covaris machine (Imperial College London) with the manufacturer's recommended settings to achieve ~400bp fragment peaks. Fragment distribution was assessed using an Agilent TapeStation 4150 using a dsDNA D1000 or D5000 cartridge. Final libraries were QC before shipment by Qubit dsDNA assay and TapeStation as above. Libraries were submitted for sequencing to Novogene(UK)
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Obtaining a complete picture of 3D-genome organization in tissues and disease states is often blocked by limiting amounts of tissue samples such that multiple types of genome-wide analyses cannot be performed on the same sample. Here, we found that starting with a pure population of memory CD4+ T-cells (mCD4-cells) we could get reproducible Hi-C data with just 50,000 cells. This population is only ~18.75% of the whole T-cell population, but presumably its uniformity enabled high reproducibility with low cell numbers so that Hi-C, RNA-Seq and potentially other analyses could be performed with repeats from the same donor. Moreover, there was strong correspondence between data from 3 different donors, suggesting that variability between individuals observed in other reports may reflect more the dynamic variability in the status of the immune system between individuals rather than variability in genome organization between individuals for a given cell type. We further observed uniform changes between samples and blood donors occurring during activation of the memory mCD4+ T cell population. While some genome organization changes occurred concomitant with changes in gene expression, there were at least as many changes that occurred without corresponding changes in expression. Counter to the hypothesis that TADs are largely invariant structures providing a scaffold for dynamic looping contacts between enhancers and promotors, we found that there were at least as many dynamic TAD changes as loops in this highly differentiated cell type. Stimulation with IL-2 alone triggered many changes in genome organization and many of these changes were strengthened by additional TCR and CD28 co-receptor stimulation. This suggests a stepwise process whereby mCD4+ T cells undergo sequential genome organization changes induced by distinct or combined stimuli likely to "prime" or “de-prime” them for expression responses to subsequent TCR-antigen ligation or additional cytokine stimulation Overall design: Primary CD4+ memory T-cells were isolated from three blood donors and purified via FACS. Cells were treated with either IL-2, IL2+TCR or left untreated. HiC experiments were performed on 'donor1' in duplicate using either 50000, 250000 or 1000000 cells in order to compare the reliability of the method using reduced numbers of input cells. For donors 2 and 3, 250000 cells were employed in single experiments.
SAMPLE_TITLE: donor1, IL2, 250000 cells, replicate 2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "blood"}, {"TAG": "tissue", "VALUE": "blood"}, {"TAG": "cell type", "VALUE": "primary CD4+ memory T-cells"}, {"TAG": "donor", "VALUE": "donor1"}, {"TAG": "treatment", "VALUE": "IL2 activation"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: donor1, IL2, 250000 cells, replicate 2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: donor1, IL2, 250000 cells, replicate 2
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