EXPERIMENT_TITLE: GSM4887843: IMR90_rep4; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Cells were fixed with 1% (v/v) formaldehyde in PBS for 10 min at room temperature. Fixation was quenched 0.2M glycene. Cells were washed with cold PBS twice and pelleted. Cell pellet was resuspended in a premixture of 250 µl cold lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630) and 50 µl protease inhibitors. The crude nuclei were permeabilized by resuspending in 200 µl of 0.5% Triton X-100 in lysis buffer and incubating at room temperature for 15 min. To carry out triple digestion, the nulei were then pelleted by centrifugation and resuspended in 250 µl of master mix containing 0.1% SDS, 1x Cutsmart buffer (NEB), 10 µl MboI (NEB R0147S), 5 µl CviQI (NEB R0639S), and 5 µl CviAII (NEB R0640S). The digestion was performed at room temperature for 2 hours, and then 37 oC for 2 hours. The restriction enzymes were then inactivated by incubating at 65 oC for 20 min. After cooling to room temperature, end blunting was performed by adding a 50 µl master mix containing 1.5 µl of 10 mM dCTP, 1.5 µl of 10 mM dCTP, 1.5 µl of 10 mM dCTP, 37.5 µl of 0.4 mM biotin-dATP (ThermoFisher), and 4 µl of DNA polymerase I Klenow (NEB), and incubating at 37 oC for 1 hour with rotation. For blunt end ligation, the suspension was then mixed with 200 µl of master mix containing 50 µl 10x T4 ligase buffer (NEB), 2.5 µl 20 mg/ml BSA, and 5 µl of 400 U/µl T4 ligase (NEB). To maximize the ligation efficiency for the low input Tri-HiC protocol, we alternatated the temperature condition by first incubating the mixture for 2 hours at room temperature,then overnight at 4 oC, and lastly 2 additional hours at room temperature. The processed nuclei were pelleted and resuspended in 500 µl 1x T4 ligation buffer supplemented with 50 µl 20 mg/ml proteinase K and 50 µl 10% SDS, and incubated at 55 oC for 30 min. For de-crosslinking, 60 µl 5M sodium chloride was added and the mixture was incubated at 68 oC for 2 hours or orvernight. The DNA content was then purified by phenol-chloroform extraction, as described in the Tri-4C protocol, and resuspended in 15-50 µl 10 mM Tris-HCl, scaling with the input. To construct Tri-HiC Illumina library, we performed the following reactions defined in units. Each unit started with 300 ng purified proximity-ligated DNA quantified by Qubit (ThermoFisher), which should be obtained from < 100k starting cells. For tagmentation, one unit of input was diluted to 30 µl and mixed with 50 µl of 2x TD buffer and 20 µl of TDE (Illumina 20034197, Lot 20436911), and incubated at 55 oC for 10 min (Note: the amount of TDE depends on the storage condition of the enzyme. We recommend optimizing the exact amount to obtain fragments peaked at 250-400 bp range). The tagmented DNA was purified with DNA Clean & Concentrator-5 (Zymo Research) and eluted in 30 µl elution buffer. The eluent was mixed with 50 µl 2x NEBNext high fidelity PCR master mix, 1.25 µM ATAC-seq primers, and diluted to 100 µl final volume to be amplified by a 2-cycle PCR with the cycling condition of 72 oC for 5 min, 2 cycles of 98 oC for 10 s, 63 oC for 10 s, 72 oC for 1 min, and final extension at 72 oC for 5 min. Meanwhile, 15 µl of 10mg/mL Streptavidin C1 Dynabeads (ThermoFisher) was prepared by washing 3 times with 200 µl of 2x Binding and washing (B&W) buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl). The beads were resuspended with 100 µl 2x B&W buffer and were diretly added to the PCR product, mixed with pipetting, and incubated for 15 min at room temperature with rotation. Beads were then separated on a magnet and washed 3 times with 500 µl 1x B&W buffer (5 min incubation each), 1 time with 10 mM Tris-HCl, and finally resuspended in 100 µl PCR solution containing 50 µl 2x NEBNext high fidelity PCR master mix and 0.5 µM ATAC-seq primers. A following PCR reaction was performed with the cycling condition of 98 oC for 30 sec, 8 cycles of 98 oC for 10 s, 63 oC for 10 s, 72 oC for 1 min, and final extension at 72 oC for 5 min (Note: we recommend to remove beads from the reaction at the end of the 4th cycle). The amplified library was size-selected and purified by 0.55-1x SPRI beads before QC and sequencing. Tri-HiC
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The resolution limit of chromatin conformation capture methodologies (3Cs) has restrained their application in detection of fine-level chromatin structure mediated by cis-regulatory elements (CREs). Here we report two 3C-derived methods, Tri-4C and Tri-HiC, utilizing mult-restriction enzyme digestions capable of achieveing ultrafine genome digestion for targeted and global chromatin interaction mapping, respectively, at a one hundred basepair resolution. Tri-4C identified loops associated with CREs including those involving regulatory elements devoid of typical epigenomic marks, quantifatively revealing alterations in interaction networks underlying dynamic gene control. Tri-HiC successfully uncovers fine-gage enhancer:promoter (E:P) loops at genome-wide level, identifying > 20-fold more CRE loops than in situ HiC. In addition to refined loop detection, Tri-HiC also reveals interaction stripes and contact domain insulation from promoters and enhancers, revealing their loop extrusion behaviors resembling the topologically-associated domain (TAD) boundaries. Tri-4C and Tri-HiC provide robust approaches to achieve the high resolution interactome maps required for characterizing fine-gage regulatory chromatin interactions in analysis of development, homeostasis and disease. Overall design: High resolution mapping of global long range chromatin interaction in IMR90 cells
SAMPLE_TITLE: IMR90_rep4
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Lung"}, {"TAG": "cell type", "VALUE": "Lung Fibroblast"}, {"TAG": "passages", "VALUE": "27"}, {"TAG": "cell line", "VALUE": "IMR90"}, {"TAG": "digestion", "VALUE": "Triple"}]
Pool_Member_@attributes_sample_title: IMR90_rep4
RUN_SET_RUN_Pool_Member_@attributes_sample_title: IMR90_rep4
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR13001679.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos3/sra-pub-zq-22/SRR013/13001/SRR13001679/SRR13001679.lite.1", "size": "156063616119", "date": "2022-09-11 17:01:39", "md5": "1300056fd31a4a864ef19af4a9988c74", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR13001679", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR13001679/SRR13001679", "size": "200199356327", "date": "2021-04-07 19:35:33", "md5": "7ba10fc925b9c0c215296c39228d9ce4", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}]