EXPERIMENT_TITLE: HiSeq X Ten paired end sequencing; The chromosomal architecture of acute myelocytic leukemia HL-60 cells upon all-trans retinoid acid treatment
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: HL60-cells were treated with ATRA for 2 and 5 days, and harvested for Hi-C study. 2×10^7 Cells were fixed by 1% formaldehyde (v/v) for 15 min at room temperature (RT) with slowly rotation. Cells were lysed by 550 μl lysis buffer (500 μl 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630 and 50 μl protease inhibitors) using a homogenizer, and harvested the chromatin at 5,000 rpm and washed twice. The pellet was added 1% SDS and incubated at 65 oC for 10 min, then quenched by Triton X-100. The chromatin was digested by 600 U MboI (NEB, Ipswich, USA) at 37 oC overnight. For 3C, HindIII and KpnI were used to digeste the chromatin. The next day, enzymes were inactivated by 10% SDS at 65 oC for 30 min, and the restriction fragment overhangs were filled in using biotin-14-dCTP and Klenow at 37 oC for 45 min, and the ligation was conducted in 7.61 ml ligation mix (745 μl 10% Triton X-100, 745 μl 10x ligation buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 100 mM DTT), 80 μl 10 mg/ml BSA, 80 μl 100 mM ATP and 5.96 ml water) for 4 h at 16°C. The blunt-end Hi-C ligation was sonicated and pull down by streptavidin beads, then purified by ethanol. Hi-C library was prepared for paired ending sequencing using NEBNext® UltraTM DNA Library Prep Kit for Illuina® (NEB).
STUDY_DESCRIPTOR_STUDY_ABSTRACT: 2×10^7 HL60 cells were treated with 10 uM ATRA for 2 and 5 days, and conducted Hi-C to study the chromosomal architecture. Cells were fixed by 1% formaldehyde (v/v) for 15 min at room temperature (RT) with slowly rotation. Cells were lysed by 550 µl lysis buffer (500 µl 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630 and 50 µl protease inhibitors) using a homogenizer, and harvested the chromatin at 5,000 rpm and washed twice. The pellet was added 1% SDS and incubated at 65 oC for 10 min, then quenched by Triton X-100. The chromatin was digested by 600 U MboI (NEB, Ipswich, USA) at 37 oC overnight.  The next day, enzymes were inactivated by 10% SDS at 65 oC for 30 min, and the restriction fragment overhangs were filled in using biotin-14-dCTP and Klenow at 37 oC for 45 min, and the ligation was conducted in 7.61 ml ligation mix (745 µl 10% Triton X-100, 745 µl 10x ligation buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 100 mM DTT), 80 µl 10 mg/ml BSA, 80 µl 100 mM ATP and 5.96 ml water) for 4 h at 16°C. The blunt-end Hi-C ligation was sonicated and pull down by streptavidin beads, then purified by ethanol. Hi-C library was prepared for paired ending sequencing using NEBNext® UltraTM DNA Library Prep Kit for Illuina® (NEB). For regular 3C ligation, after enzymes inactivation, 10 µl 1 U/µl T4 DNA ligase was added to incubate at 16°C overnight. The interaction was detected by qPCR using the specific 3C primers (Table 1) followed by DNA purification. Hi-C was conducted in PE-150. An iterative method for Hi-C mapping with initial length 30bp, if it fails to map uniquely, the next round of mapping for additional 20 bp continued, this procedure lasts until the full read length reaches 150bp. The Hi-C reads were mapped to the human reference genome (assembly GRCh38) using bowtie2 (v2.2.9). The reads mapped to the same restriction fragment, the reads less than 500bp and PCR duplicates were all removed. Hi-C contact maps were normalized using ICE.
SAMPLE_TITLE: HL60_d5
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "ENA-FIRST-PUBLIC", "VALUE": "2020-05-07T17:05:44Z"}, {"TAG": "ENA-LAST-UPDATE", "VALUE": "2019-11-25T14:21:51Z"}, {"TAG": "External Id", "VALUE": "SAMEA6245498"}, {"TAG": "INSDC center name", "VALUE": "The Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou, Henan 450008, PR. China"}, {"TAG": "INSDC first public", "VALUE": "2020-05-07T17:05:44Z"}, {"TAG": "INSDC last update", "VALUE": "2019-11-25T14:21:51Z"}, {"TAG": "INSDC status", "VALUE": "public"}, {"TAG": "Submitter Id", "VALUE": "E-MTAB-8547:HL60_d5"}, {"TAG": "broker name", "VALUE": "ArrayExpress"}, {"TAG": "cell line", "VALUE": "HL-60"}, {"TAG": "cell type", "VALUE": "leukocyte"}, {"TAG": "common name", "VALUE": "human"}, {"TAG": "developmental stage", "VALUE": "adult"}, {"TAG": "disease", "VALUE": "acute promyelocytic leukemia"}, {"TAG": "genotype", "VALUE": "wild type genotype"}, {"TAG": "organism part", "VALUE": "blood"}, {"TAG": "sample name", "VALUE": "E-MTAB-8547:HL60_d5"}, {"TAG": "scientific_name", "VALUE": "Homo sapiens"}]
Pool_Member_@attributes_sample_title: HL60_d5
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HL60_d5
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "ERR3680685.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos2/sra-pub-zq-18/ERR003/3680/ERR3680685/ERR3680685.lite.1", "size": "38409046094", "date": "2022-08-14 05:54:01", "md5": "42f53c96e82deae75499cff65c2df494", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "ERR3680685", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/ERR3680685/ERR3680685", "size": "58485784106", "date": "2022-07-17 09:26:11", "md5": "e272b2e2f190a7488262376fac799759", "version": "2", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HL60_d5_R2.fq.gz", "size": "41076092267", "date": "2022-07-17 09:06:34", "md5": "89cb13d12b6721ac4c218ab8ceafb75b", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HL60_d5_R1.fq.gz", "size": "38349305272", "date": "2022-07-17 08:58:46", "md5": "c3b09c11b47ff5a690b2f542c8a00841", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]