EXPERIMENT_TITLE: GSM4955380: HiC-A549-8h-rep1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Twenty-four hours post infection, media was removed from the well, and replaced with Fixation buffer (PBS, 2% FBS, 1% Methanol-Free Formaldehyde). Cells were fixed at room temperature for 10 min. 2M Glycine was then added to a final concentration of 0.125M, and cells were incubated at room temperature for 5min to quench the reaction. Supernatants were then removed wells, and each well was washed 2 times with cold PBS. Cells were then lysed in the well using 250 µl of Lysis Buffer [0.5% SDS + Halt Protease and Phosphatase Inhibitors (Thermo Scientific)]. Cell lysates were collected in a 1.5ml tube. 1.5 mU RNAseA (Thermo Scientific) was added to each lysate, and lysates were then incubated at 37oC for 1h. RNAseA treated lysates were then snap frozen and stored in -80°C. After thawing, nuclei were collected at 1500 g for 5 minutes at room temperature. Most of the supernatant was discarded, leaving the nuclei in 10 µl liquid. Samples were resuspended in reaction buffer (25 µl 10% Triton X-100, 25 µl 10x Dpn II buffer, 188 µl water) and rotated at 37°C, 8 RPM for 15 minutes. Chromatin was digested overnight (ON) with either 2 µl (100 U) Dpn II (NEB) (later experiments) at 37°C, rotating overhead with 8 RPM. Nuclei were collected by centrifugation at 1500 g for 5 minutes at room temperature, 225 µl of the supernatant were discarded, leaving the nuclei in 25 µl liquid, and overhangs were filled in with Biotin-14-dATP by adding 75 µl Klenow Master Mix (54.45 µl water, 7.5 µl NEBuffer 2, 0.35 µl 10 mM dCTP, 0.35 µl 10 mM dTTP, 0.35 µl 10 mM dGTP, 7.5 µl 0.4 mM Biotin-14-dATP (Invitrogen), 2 µl 10% Triton X-100, 2.5 µl (12.5 U) Klenow fragment (Enzymatics)) and rotating overhead at RT, 8 RPM for 40 minutes. Reactions were stopped by adding 2.5 µl 0.5 M EDTA and placed on ice. Proximity ligation was performed by transferring the entire reaction into 1.5 ml Eppendorf tubes containing 400 µl ligase mix (322.5 µl water, 40 µl 10x T4 DNA ligase buffer (Enzymatics), 36 µl 10% Triton X-100, 0.5 µl 10 % BSA, 1 µl (600 U) T4 DNA ligase (HC, Enzymatics) and rotating ON at 16°C, 8 RPM. Reactions were stopped with 20 µl 0.5 M EDTA, treated with 1 µl 10 mg/ml DNase-free RNAse A for 15 minutes at 42°C, then 31 µl 5 M NaCl, 29 µl 10 % SDS and 5 µl 20 mg/ml DNase-free proteinase K (Thermo) were added, proteins digested for 1 h at 55°C while shaking at 800 RPM, then crosslinks reversed ON at 65°C. After extraction with 600 µl pH 8-buffered phenol/chloroform/isoamyl alcohol (Ambion) followed by extraction with 600 µl chloroform, DNA was precipitated with 1.5 µl (22.5 µg) Glycoblue (Ambion) and 1400 µl 100% ethanol ON at -20°C. After centrifugation for 20' at 16000g, 4°C, the DNA pellet was washed twice with 80% ethanol, and the pellet air-dried and dissolved in 131 µl TT (0.05% Tween 20/Tris pH 8). DNA (200 ng) was sheared to 300 bp in 130 µl TT on a Covaris E220 sonicator using the manufacturer's protocol. Biotinylated DNA was captured on Dynabeads MyOne Streptavidin T1 (Thermo) by combining the sonicated DNA sample (130 µl) with 20 µl Dynabeads that had previously been washed with 1x B&W buffer (2X B&W: 10 mM Tris-HCl pH=7.5, 1 mM EDTA, 2 M NaCl) and had been resuspended in 130 µl 2x B&W containing 0.2% Tween 20. The binding reaction was rotated at RT for 45 minutes, and DNA-bound beads were vigorously washed twice with 150 µl 1x B&W/0.1% Triton-X 100, once with 180 µl TET (0.05% Tween 20, 10 mM Tris pH 8, 1 mM EDTA). Libraries were prepared on-beads using an NEBnext Ultra II DNA library prep kit using half the reagent/reaction volumes given in NEB's instruction manual and 1.6 pmol Bioo DNA sequencing adapters (Illumina TruSeq-compatible) per reaction. Reactions were stopped by adding 5 µl 0.5 M EDTA, beads collected on a magnet and washed twice with 150 µl 1x B&W/0.1% Triton-X 100, twice with 180 µl TET and resuspended in 20 µl TT (0.05% Tween 20, 10 mM Tris pH 8.0). Libraries were amplified by PCR for 10 cycles (98°C, 30s; 10x [98°C, 10s; 63°C, 20s; 72°C, 30s]; 72°C, 2 min; 4°C, ∞), using 10 µl of the bead suspension in a 50 µl reaction with NEBNext Ultra II Q5 2x master mix (NEB) and 0.5 µM each Solexa 1GA/1GB primers (Solexa 1GA: AATGATACGGCGACCACCGA, Solexa 1GB: CAAGCAGAAGACGGCATACGA). Libraries were precipitated onto magnetic beads by adding 40 µl 20% PEG8000/2.5 M NaCl and 2 µl SpeedBeads (8.9 % PEG final) to 48 µl PCR reaction, thorough mixing by vortexing followed by 10-minute incubation at RT. Beads were collected using a magnet and the supernatant discarded. After washing beads twice by adding 180 µl 80% EtOH, moving the tube strip 6x from side to side of the magnet, collecting beads and discarding the supernatant, beads were air-dried, and DNA eluted by adding 20 µl TT. Libraries were sequenced paired-end for 42 cycles each to a depth of approximately 250 million reads per experiment on an Illumina NextSeq 500 sequencer.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Targeting these pathways might therefore be a viable therapeutic strategy. Previously, we have reported that chromatin factors such as Topoisomerase I (Top1) play key roles in controlling the induction of inflammatory gene expression programs. Here, by using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we show that Topoisomerase 1 (Top1) inhibition in infected cells and animals suppresses lethal inflammation induced by SARS-CoV-2. Overall design: To study alterations in genome structure after SARS-CoV-2 infection, we performed Hi-C on SARS-CoV-2 infected A549-ACE2 cells at 0,8 and 24 hours post infection. Experiment was done in biological duplicate. Each biological replicate was sequenced with 4 technical replicates. To study the impact of Top1 on the inflammatory response to SARS-CoV-2, we knocked-down TOP1 expression using siRNA in A549-ACE2 cells. Cells were then left uninfected or infected with SARS-CoV-2. Cells that were not treated with siRNA (no siRNA) or scrambled siRNA (siSCR) were used as controls. The experiment was done in triplicate.
SAMPLE_TITLE: HiC-A549-8h-rep1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "A549-ACE2"}, {"TAG": "cell line", "VALUE": "A549-ACE2"}, {"TAG": "infection", "VALUE": "Infected"}, {"TAG": "sirna", "VALUE": "NA"}, {"TAG": "time point", "VALUE": "8h"}]
Pool_Member_@attributes_sample_title: HiC-A549-8h-rep1
RUN_SET_RUN: [{"IDENTIFIERS": {"PRIMARY_ID": "SRR13192959", "SUBMITTER_ID": "GSM4955380_r1"}, "EXPERIMENT_REF": null, "Pool": {"Member": {"IDENTIFIERS": {"PRIMARY_ID": "SRS7828287", "EXTERNAL_ID": ["SAMN16988745", "GSM4955380"]}, "@attributes": {"member_name": "", "accession": "SRS7828287", "sample_name": "GSM4955380", "sample_title": "HiC-A549-8h-rep1", "spots": "41269418", "bases": "3466631112", "tax_id": "9606", "organism": "Homo sapiens"}}}, "SRAFiles": {"SRAFile": [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR13192959.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos3/sra-pub-zq-24/SRR013/13192/SRR13192959/SRR13192959.lite.1", "size": "872177099", "date": "2022-08-03 01:19:39", "md5": "51f0b19875603a19000c37e3c8ba1a6c", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR13192959", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR13192959/SRR13192959", "size": "1277388335", "date": "2020-12-03 14:17:46", "md5": "c55e5605aa624bcd47bd0fa7a49ee8aa", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HiC-A549-08h-r1-t1_R2.fastq.gz", "size": "1264656635", "date": "2020-12-03 14:17:08", "md5": "cf85746f11fe5ea46cde95423bb0a021", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HiC-A549-08h-r1-t1_R1.fastq.gz", "size": "1261745313", "date": "2020-12-03 14:16:40", "md5": "589cd109f51e92ebc7d828dbb0765291", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]}, "CloudFiles": {"CloudFile": [null, null, null, null]}, "Statistics": {"Read": [null, null], "@attributes": {"nreads": "2", "nspots": "41269418"}}, "Databases": {"Database": {"Table": {"Statistics": {"Rows": null, "Elements": null, "@attributes": {"source": "meta"}}, "@attributes": {"name": "SEQUENCE"}}}}, "Bases": {"Base": [null, null, null, null, null], "@attributes": {"cs_native": "false", "count": "3466631112"}}, "@attributes": {"alias": "GSM4955380_r1", "accession": "SRR13192959", "total_spots": "41269418", "total_bases": "3466631112", "size": "1277386805", "load_done": "true", "published": "2021-03-30 19:38:37", "is_public": "true", "cluster_name": "public", "has_taxanalysis": "1", "static_data_available": "1"}}, {"IDENTIFIERS": {"PRIMARY_ID": "SRR13192960", "SUBMITTER_ID": "GSM4955380_r2"}, "EXPERIMENT_REF": null, "Pool": {"Member": {"IDENTIFIERS": {"PRIMARY_ID": "SRS7828287", "EXTERNAL_ID": ["SAMN16988745", "GSM4955380"]}, "@attributes": {"member_name": "", "accession": "SRS7828287", "sample_name": "GSM4955380", "sample_title": "HiC-A549-8h-rep1", "spots": "43553792", "bases": "3658518528", "tax_id": "9606", "organism": "Homo sapiens"}}}, "SRAFiles": {"SRAFile": [{"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HiC-A549-08h-r1-t2_R1.fastq.gz", "size": "1410977029", "date": "2020-12-03 14:19:25", "md5": "c8aa2ca017bf0512a571819bed5fed71", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HiC-A549-08h-r1-t2_R2.fastq.gz", "size": "1407825638", "date": "2020-12-03 14:18:51", "md5": "5f566469ffe3e82b2f423bc5c9e9cd67", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR13192960", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR13192960/SRR13192960", "size": "1446698961", "date": "2020-12-03 14:20:29", "md5": "134d85ea1a02f8f2b056fdd1381a654c", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR13192960.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos3/sra-pub-zq-22/SRR013/13192/SRR13192960/SRR13192960.lite.1", "size": "922185809", "date": "2022-07-21 00:40:04", "md5": "0adbb45a0bbe3c734ff28ea24f01239d", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}]}, "CloudFiles": {"CloudFile": [null, null, null, null]}, "Statistics": {"Read": [null, null], "@attributes": {"nreads": "2", "nspots": "43553792"}}, "Databases": {"Database": {"Table": {"Statistics": {"Rows": null, "Elements": null, "@attributes": {"source": "meta"}}, "@attributes": {"name": "SEQUENCE"}}}}, "Bases": {"Base": [null, null, null, null, null], "@attributes": {"cs_native": "false", "count": "3658518528"}}, "@attributes": {"alias": "GSM4955380_r2", "accession": "SRR13192960", "total_spots": "43553792", "total_bases": "3658518528", "size": "1446697432", "load_done": "true", "published": "2021-03-30 19:38:37", "is_public": "true", "cluster_name": "public", "has_taxanalysis": "1", "static_data_available": "1"}}, {"IDENTIFIERS": {"PRIMARY_ID": "SRR13192961", "SUBMITTER_ID": "GSM4955380_r3"}, "EXPERIMENT_REF": null, "Pool": {"Member": {"IDENTIFIERS": {"PRIMARY_ID": "SRS7828287", "EXTERNAL_ID": ["SAMN16988745", "GSM4955380"]}, "@attributes": {"member_name": "", "accession": "SRS7828287", "sample_name": "GSM4955380", "sample_title": "HiC-A549-8h-rep1", "spots": "38897687", "bases": "3267405708", "tax_id": "9606", "organism": "Homo sapiens"}}}, "SRAFiles": {"SRAFile": [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR13192961.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos3/sra-pub-zq-24/SRR013/13192/SRR13192961/SRR13192961.lite.1", "size": "824364671", "date": "2022-08-03 01:19:50", "md5": "09a1abd928c6626606cb872e0b50ae67", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR13192961", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR13192961/SRR13192961", "size": "1314325655", "date": "2020-12-03 14:18:17", "md5": "a465f73019dabe1b1c1dab6731f32ddf", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HiC-A549-08h-r1-t3_R2.fastq.gz", "size": "1280448216", "date": "2020-12-03 14:17:23", "md5": "6b6adcc4c2b445e7a19cfb73f0eea7f0", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HiC-A549-08h-r1-t3_R1.fastq.gz", "size": "1276578228", "date": "2020-12-03 14:16:58", "md5": "4a07544501600c148bf3fa5a64f6d066", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]}, "CloudFiles": {"CloudFile": [null, null, null, null]}, "Statistics": {"Read": [null, null], "@attributes": {"nreads": "2", "nspots": "38897687"}}, "Databases": {"Database": {"Table": {"Statistics": {"Rows": null, "Elements": null, "@attributes": {"source": "meta"}}, "@attributes": {"name": "SEQUENCE"}}}}, "Bases": {"Base": [null, null, null, null, null], "@attributes": {"cs_native": "false", "count": "3267405708"}}, "@attributes": {"alias": "GSM4955380_r3", "accession": "SRR13192961", "total_spots": "38897687", "total_bases": "3267405708", "size": "1314324126", "load_done": "true", "published": "2021-03-30 19:38:37", "is_public": "true", "cluster_name": "public", "has_taxanalysis": "1", "static_data_available": "1"}}, {"IDENTIFIERS": {"PRIMARY_ID": "SRR13192962", "SUBMITTER_ID": "GSM4955380_r4"}, "EXPERIMENT_REF": null, "Pool": {"Member": {"IDENTIFIERS": {"PRIMARY_ID": "SRS7828287", "EXTERNAL_ID": ["SAMN16988745", "GSM4955380"]}, "@attributes": {"member_name": "", "accession": "SRS7828287", "sample_name": "GSM4955380", "sample_title": "HiC-A549-8h-rep1", "spots": "74190648", "bases": "6232014432", "tax_id": "9606", "organism": "Homo sapiens"}}}, "SRAFiles": {"SRAFile": [{"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HiC-A549-08h-r1-t4_R1.fastq.gz", "size": "2311979183", "date": "2020-12-03 14:36:58", "md5": "f5b9e8d72ace33647183757f28c8eb59", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HiC-A549-08h-r1-t4_R2.fastq.gz", "size": "2323998094", "date": "2020-12-03 14:37:40", "md5": "a3c7af786e9dd2ff90087226ffe54857", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR13192962", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR13192962/SRR13192962", "size": "2373356950", "date": "2020-12-03 14:38:38", "md5": "b63f660d03557671d33d051aa98648ed", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR13192962.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos2/sra-pub-zq-20/SRR013/13192/SRR13192962/SRR13192962.lite.1", "size": "1567443526", "date": "2022-08-01 01:49:08", "md5": "5d7fb84e11a456d50f25db44526773a9", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}]}, "CloudFiles": {"CloudFile": [null, null, null, null]}, "Statistics": {"Read": [null, null], "@attributes": {"nreads": "2", "nspots": "74190648"}}, "Databases": {"Database": {"Table": {"Statistics": {"Rows": null, "Elements": null, "@attributes": {"source": "meta"}}, "@attributes": {"name": "SEQUENCE"}}}}, "Bases": {"Base": [null, null, null, null, null], "@attributes": {"cs_native": "false", "count": "6232014432"}}, "@attributes": {"alias": "GSM4955380_r4", "accession": "SRR13192962", "total_spots": "74190648", "total_bases": "6232014432", "size": "2373355419", "load_done": "true", "published": "2021-03-30 19:38:37", "is_public": "true", "cluster_name": "public", "has_taxanalysis": "1", "static_data_available": "1"}}]