EXPERIMENT_TITLE: GSM6234769: Hi-C, MCF7, 2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Three million cells were fixed with freshly made 1% formaldehyde for 10 min at room temperature for each sample. The reaction was quenched by adding 2 M glycine to a final concentration of 0.2 M for 5 min at room temperature. Fixed cells were lysed in lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl and 0.2% IGEPAL CA-630 with proteinase inhibitor) for 1h at 4 °C with rotation. The supernatant was discarded and the pellet was gently resuspended in 1 × CutSmart Buffer (NEB, B7204) containing 0.1% SDS and incubated at 65°C for 10 min. Permeabilization was quenched by adding 10% Triton X-100 and incubated at 37°C for 1h. Chromatin was digested overnight at 37 °C with rotation by adding the DpnII restriction enzyme (NEB, R0543M). Digested chromatin ends were filled and marked with biotin-14-dATP (Thermo Fisher, 19524016) using a large Klenow fragment of DNA polymerase I (NEB, M0210) for 1h at 37 °C. Samples were then diluted and ligated using T4 DNA ligase (Thermo Fisher, EL0013) at 16 °C overnight. Chromatin was de-cross-linked, and proteins were removed by proteinase K, followed by phenol/chloroform extraction coupled with isopropanol precipitation using glycogen as a carrier. Biotin-14-dATP from unligated DNA ends was removed by T4 DNA polymerase (NEB, M0203) at 12 °C for 2 h. DNA was purified by phenol/chloroform and sonicated using a Covaris S220 (Covaris, USA) to approximately 300-600 bp. Biotinylated DNA fragments were then pulled down using Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher, 65001). The DNA ends were subsequently repaired and phosphorylated using T4 DNA polymerase (NEB, M0203), Klenow fragment of DNA polymerase I (NEB, M0210), and T4 Polynucleotide Kinase NK (NEB, M0201). The DNA was further prepared for sequencing using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme Biotech, ND607) and barcoded adaptors were ligated using TruePrep Index Kit V2 for Illumina (Vazyme Biotech, TD202). Each Hi-C library was indexed uniquely and amplified by 12 cycles of PCR for all samples. PCR purification and size selection was carried out using the VAHTS DNA Clean Beads (Vazyme Biotech, N411). Library quantity was assessed using a Qubit 3.0 Fluorometer (Invitrogen, USA). For each individual, the library was sequenced with 150 bp paired-end reads on the Illumina HiSeq X Ten platform.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Doxorubicin resistance remains a major therapeutic challenge leading to treatment failure and poor survival prognosis in breast cancer. Although doxorubicin induces massive changes in the transcriptional landscape accompanied by alterations of chromatin accessibility are well known, potential diagnostic or therapeutic targets associated with three-dimensional (3D) genome reorganization in doxorubicin-resistant breast cancer cells have not yet been systematically investigated. Here we performed integrated analyses combining in situ high-throughput chromosome conformation capture (Hi-C), ATAC-seq and mHi-C data on doxorubicin-resistant MCF7 human breast cancer cells compared to parental cells. It revealed that A/B compartment switching was positively correlated to genome-wide differential gene expression, and the genome was spatially reorganized into smaller topologically associating domains (TADs) and loops. We also revealed the contribution of increased chromatin accessibility and potential transcription factor families, including CTCF and BORIS, to gained TAD boundaries and loop anchors. Intriguingly, we observed two condensed genomic regions (~20kb) with decreased chromatin accessibility flanking TAD boundaries, which might play a critical role in the formation or maintenance of TADs. Moreover, we identified a number of gained and lost enhancer-promoter interactions associated with differentially expressed genes, including FA2H, FOXA1, JRKL and EZH, some of which involved in chromatin organization and breast cancer signaling pathways. This study uncovered a close connection between 3D genome reorganization, chromatin accessibility as well as gene transcription, and provides resources and novel insights into the epigenomic mechanisms with potential therapeutic implications for doxorubicin resistance in breast cancer. Overall design: Genome-wide landscaps of 3D chromatin architecture in parental and doxorubicin-resistant breast cancer cells. In total, 4 samples were analyzed. Hi-C_MCF7_1 and Hi-C_MCF7_2 were two biological replicates and served as the control group. Hi-C_MCF7-DR_1 and Hi-C_MCF7-DR_2 were two biological replicates and served as the treatment group.
SAMPLE_TITLE: Hi-C, MCF7, 2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Breast cancer cell line"}, {"TAG": "cell line", "VALUE": "MCF7 cell line"}, {"TAG": "treatment", "VALUE": "untreated"}, {"TAG": "phenotype", "VALUE": "parental"}]
Pool_Member_@attributes_sample_title: Hi-C, MCF7, 2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: Hi-C, MCF7, 2
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR19627182.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos1/sra-pub-zq-38/SRR019/19627/SRR19627182/SRR19627182.lite.1", "size": "34965050258", "date": "2022-09-15 04:33:15", "md5": "32a2f986d017c3cecd07526bf049d5d9", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR19627182", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR19627182/SRR19627182", "size": "48151323234", "date": "2022-06-11 05:24:24", "md5": "68daee0c8046f02d50504d06679f2372", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "Hi-C_MCF7_2_R2.fastq.gz", "size": "34645823626", "date": "2022-06-11 05:05:48", "md5": "9b0cf06f5f43aba20ad707442c279c77", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "Hi-C_MCF7_2_R1.fastq.gz", "size": "32936432441", "date": "2022-06-11 05:03:45", "md5": "34a8567c6b1236b3fb1adab28b071990", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]