EXPERIMENT_TITLE: GSM3395121: T47D_Hi-C_noSerum_siGTF3C5_rep2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: In situ Hi-C library preparationIn situ Hi-C was performed as previously described (Rao et al., 2014) with the following modifications: (i) 2x106 cells were used as starting material; (ii) chromatin was initially digested with 100 U MboI (New England BioLabs) for 2 h, and then another 100 U (2 h incubation) and a final 100 U were added before overnight incubation; (iii) before fill-in with bio-dATP, nuclei were pelleted and resuspended in fresh 1× NEB2 buffer; (iv) ligation was performed overnight at 24 °C with 10,000 cohesive end units per reaction; (v) de-cross-linked and purified DNA was sonicated to an average size of 300-400 bp with a Bioruptor Pico (Diagenode; seven cycles of 20 s on and 60 s off); (vi) DNA fragment-size selection was performed only after final library amplification; (vii) library preparation was performed with an NEBNext DNA Library Prep Kit (New England BioLabs) with 3  μl NEBNext adaptor in the ligation step; (viii) libraries were amplified for 8-12 cycles with Herculase II Fusion DNA Polymerase (Agilent) and were purified/size-selected with Agencourt AMPure XP beads (>200 bp). Hi-C library quality was assessed through ClaI digestion and low-coverage sequencing on an Illumina NextSeq500 instrument, after which every technical replicate (n = 2) of each biological replicate (n = 2) was sequenced at high coverage on an Illumina HiSeq2500 instrument. Data from technical replicates were pooled for downstream analysis. We sequenced >18 billion reads in total to obtain 0.78–1.21 billion valid interactions per time point per biological replicate.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: How repetitive elements, epigenetic modifications and architectural proteins interact ensuring proper genome expression remains poorly understood. Here we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the Activity-Dependent Neuroprotector Homeobox protein (ADNP) and located near cell cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression. Overall design: Examination of TFIIIC binding and action during cellular Serum Starvation (SS)
SAMPLE_TITLE: T47D_Hi-C_noSerum_siGTF3C5_rep2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "T47D"}, {"TAG": "cell line", "VALUE": "T47D"}, {"TAG": "media", "VALUE": "RPMI supplemented with 10% charcoal-treated (CT) FBS for 48 h and starvation was achieved by culturing cells in the absence of FBS for 16 h"}, {"TAG": "serum", "VALUE": "serum-starved"}]
Pool_Member_@attributes_sample_title: T47D_Hi-C_noSerum_siGTF3C5_rep2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: T47D_Hi-C_noSerum_siGTF3C5_rep2
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": null, "@attributes": {"cluster": "public", "filename": "8276ee842_53be2f477_read2.fastq.gz", "size": "10325124919", "date": "2020-03-12 19:02:39", "md5": "d2e353fa8ea5fb9e2bc7a5c5eea31a0a", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "8276ee842_53be2f477_read1.fastq.gz", "size": "9922318844", "date": "2020-03-12 18:36:30", "md5": "625b8c4bc71fc95a54a96a910902befc", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR7875662.sralite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos3/sra-pub-zq-22/SRR007/875/SRR7875662.sralite.1", "size": "7546735840", "date": "2021-06-15 22:31:24", "md5": "ffe9739739ef672f4770481cd0fd7a56", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR7875662", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR7875662/SRR7875662", "size": "12676080012", "date": "2018-09-19 08:40:32", "md5": "6a1c0945efff447f329cda2949faaf07", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}]