EXPERIMENT_TITLE: GSM6938983: pLHiC-G1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: For phosphorylated linker Hi-C (pLHi-C), phosphorylated linker HiChIP (pLHiChIP) and spike-in phosphorylated linker HiChIP (spLHiChIP), cells were cross-linked with 1% formaldehyde for 5 min and stopped by adding 125 mM glycine for 10 min at room temperature. Nuclei were isolated and a one-step in situ digestion and dephosphorylation took place by using DpnII and rSAP enzymes. During the nuclei isolation step, only for the spLHiChIP samples, murine NHO2A cells were added as exogenous control (spike-in) at a 1:20 cell ratio. Next, biotinylated oligos with 5' end phosphate were used with T4 ligase for the ligation step. Chromatin fragmentation was performed using the Covaris Focused Ultrasonicator M220 for 30 min per ml lysate. Samples were de-crosslinked overnight and Protein and RNA were digested with proteinase K and RNase A. DNA was isolated using ChIP DNA Clean & Concentrator (Zymo Research). Biotin pull-down was performed with MyOne Streptavidin C1 beads (Thermo Fisher Scientific) according to manufacturer's instructions. Only for pLHiChIP and spLHiChIP samples, the chromatin fragmentation was followed by an immunoprecipitation of the target protein. To this end, Dynabeads Protein A and Protein G were pre-incubated overnight with 10-15 µg antibody in the presence of 5 mg/ml BSA. Next, chromatin was added to the pre-incubated beads for at least 6h at 4°C with rotation. Beads were then washed with different TRIS buffers containing distinct salt-concentrations, EDTA and detergents, followed by wahsing with only TE-buffer. The immunoprecipitated chromatin of the pLHiChIP and spLHiChIP samples was eluted with 100mM NaHCO3 and 1% SDS for 15 min. Eluted samples were de-crosslinked overnight. DNA was isolated using ChIP DNA Clean & Concentrator (Zymo Research). Biotin pull-down was performed with MyOne Streptavidin C1 beads (Thermo Fisher Scientific) according to manufacturer's instructions. Library preparation was the same for all samples and was done on beads using the NEBNext ChIP-Sequencing Library Prep Master Mix Set for Illumina (NEB) according to manufacturer's instrcutions. The only deviation from the instructions was an additional washing step between each module using a Tris-buffer with 0.5 mM EDTA, 1 M NaCl and 0.05% Tween followed by a washing step with only TE buffer. Library quality and fragment size distribution was analyzed on a Fragment Analyzer (Advanced Analytical). Sequencing was performed in single-read or paired-end mode on a NextSeq500 or NextSeq2000 platform (Illumina). pLHi-C sequencing (Illumina NextSeq500) and (s)pLHiChIP sequencing (Illumina NextSeq500 or NextSeq2000)
STUDY_DESCRIPTOR_STUDY_ABSTRACT: MYC proteins bind to virtually all active promoters transcribed by RNA polymerase II (RNAPII), but whether they interact with the three-dimensional chromatin architecture is unknown. Here we used HiChIP sequencing of the MYCN oncoprotein and found that MYCN localizes to three-dimensional hubs formed by active promoters and enhancers. In these hubs, MYCN interacts with TFIIIC, an architectural protein complex. MYCN recruits TFIIIC to promoters when transcription elongation is inhibited, and the complex of both proteins induces premature transcription termination. Termination correlates closely with the TFIIIC-dependent removal of MYCN from promoter hubs and with corresponding alterations in the three-dimensional interactions of cohesin complexes. This limits DNA damage by removing RNAPII that stalls proximal to double-strand breaks. Binding of TFIIIC to MYCN is limited by competition with Aurora-A and this protects genes involved in mRNA processing from termination, arguing that MYCN contributes to the unusual proliferative capacity of neuroblastoma cells via removing stalled RNAPII from promoter hubs and via increasing the capacity for RNA processing. Overall design: pLHiC, pLHiChIP and spLHiChIP sequencing was done with SH-EP NMYC-ER cells where the NMYC expression can be induced with 4-OHT. For some experiments, SH-EP NMYC-ER cells were used that carry a doxycycline inducible shRNA against TF3C5. For some other experiments, SH-EP NMYC-ER cells were treated with either DRB or MLN inhibitor. The immunoprecipitation in these experiments were done for either NMYC, TF3C5 or RAD21.
SAMPLE_TITLE: pLHiC-G1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "SH-EP NMYC-ER"}, {"TAG": "cell line", "VALUE": "SH-EP NMYC-ER"}, {"TAG": "cell type", "VALUE": "Neuroblastoma"}, {"TAG": "cell cycle_synchronization", "VALUE": "G1-phase synchronized"}, {"TAG": "treatment with_inhibitor", "VALUE": "not applicable"}, {"TAG": "spike-in", "VALUE": "none"}, {"TAG": "chip antibody", "VALUE": "none"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: pLHiC-G1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: pLHiC-G1
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR23099735.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos6/sra-pub-zq-40/SRR023/23099/SRR23099735/SRR23099735.lite.1", "size": "15286439687", "date": "2023-05-11 11:40:45", "md5": "cbfac9793de09c98182b0ac4cbe0d742", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR23099735", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR23099735/SRR23099735", "size": "24381554775", "date": "2023-01-19 12:29:34", "md5": "b4bd38e39cfbca090c59550ed5244fcb", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "pLHiC-G1_R2.fastq", "size": "82912483215", "date": "2023-01-19 11:50:28", "md5": "cd4a361330ea8fd3e528f1d9d2d92167", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "pLHiC-G1_R1.fastq", "size": "82920875339", "date": "2023-01-19 12:17:04", "md5": "12bf43c66cc81e7eecea670d03911991", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]