EXPERIMENT_TITLE: GSM5724113: THP-1 HiC NTC CTL; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Briefly, 2.5 x 10^6 THP-1 cells were cross-linked with 2% formaldehyde for 10 min, stopped with glycine, washed twice with PBS and snap-frozen. The thawed cells were lysed in 20 µl lysis buffer (15 min 4°C), then incubated with 24 µl Conditioning solution (19 min 62°C) and 20 µl Stop Solution 2 (15 min 37°C). Digestion with 12 µl Enzymes A1/A2 master mix was performed by sequential incubations of 45 min 37°C, 20 min 65°C and 10 min 25°C. Then, the biotin fill-in was performed with 16 µl Enzyme B master mix (45 min RT), followed by proximity ligation with 82 µl Enzyme C master mix (15 min RT). Proteins were digested with 35.5 µl Enzyme D master mix plus 20 µl Buffer E in the sequential incubations of 30 min 55°C, 90 min 68°C and 10 min 25°C. The DNA was purified using 100 µl AMPureXP beads (Beckman Coulter). After passing the QC1 quality control, 5 µg of each sample was diluted to 100 µl with Elution Buffer supplemented by 1% SDS. Fragmentation to an average fragment size of ≈400 bp was done by 15 cycles sonication in a Bioruptor Plus (Diagenode) with 30 s ON, 30 s OFF at high intensity. Size selection was performed with 60 µl and 40 µl SPRIselect beads (Beckman Coulter). Biotin enrichment was performed with 0.9-2 µg of the size selected DNA using 50 µl Enrichment Beads for < 1 µg DNA and 100 µl for > 1 µg DNA (15 min RT). Following washes with Wash Buffer and Elution Buffer end repair and adapter ligation were performed: incubation with 20 µl Enzyme W2 master mix (10 min 37°C), washes, incubation with 50 µl Enzyme G3/G4 master mix (20 min 20°C), washes, ligation of a uniquely indexed P7 TruSeq LT Adapter (Swift Biosciences Set A Indexing Kit) in 25 µl Enzyme Y1 master mix (15 min 25°C), washes, ligation of non-indexed P5 TruSeq Universal adapter (Swift Biosciences Indexing Kit) in 50 µl Enzyme B4/B5/B6 master mix, washes, resuspension. The sufficient cycle number was determined with an initial PCR using 2 µl of the resuspended beads. The remainder of the beads was subjected to library amplification. Samples, for which 100 µl Enrichment Beads had been used, were distributed into six 50 µl PCR reactions, while those with 50 µl Enrichment Beads were run in three 50 µl reactions. PCRs were performed using the KAPA 2X HiFi HotStart Ready Mix (KAPA Biosystems), KAPA Library Amplification Primer Mix and EvaGreen (BioRad). The replicate PCR reactions were pooled, separated from the beads and purified with AMPureXP beads (Beckman Coulter). Concentrations were measured using the Qubit dsDNA HS Assay Kit (Thermofisher Scientific) and quality-controled by High Sensitivity DNA Assay on an Agilent Bioanalyzer or by D1000 ScreenTape on a TapeStation (Agilent). Sequencing was performed by Dr. Stefan Günther at Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany, on an Illumina NextSeq 500-PE100.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The aim of the project was to investigate the effect of nuclear receptor activation, particularly of the vitamin D receptor, on chromatin structure, and associated functional changes in the transcriptional response of THP-1 cells. Overall design: HiC was performed on THP-1 cells, with either control (24 h vehicle) or 24 h 100 nM 1,25(OH)2D3 treatment.
SAMPLE_TITLE: THP-1 HiC NTC CTL
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Cell line"}, {"TAG": "cell type", "VALUE": "Monocyte"}, {"TAG": "cell line", "VALUE": "THP-1"}, {"TAG": "treatment", "VALUE": "control"}]
Pool_Member_@attributes_sample_title: THP-1 HiC NTC CTL
RUN_SET_RUN_Pool_Member_@attributes_sample_title: THP-1 HiC NTC CTL
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": null, "@attributes": {"cluster": "public", "filename": "Seuter_NTC_E24_R1.fastq.gz", "size": "3054831500", "date": "2021-12-08 13:28:14", "md5": "e78e1712358ee85af6b2734d323a9c54", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "Seuter_NTC_E24_R2.fastq.gz", "size": "3076716134", "date": "2021-12-08 13:27:12", "md5": "1d6b35dafd261270cc7cab0c371afd32", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR17169288", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR17169288/SRR17169288", "size": "3272797647", "date": "2021-12-08 13:30:22", "md5": "85b44d09457079af736eea76f1ca0680", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR17169288.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos2/sra-pub-zq-20/SRR017/17169/SRR17169288/SRR17169288.lite.1", "size": "2603654251", "date": "2022-09-14 08:21:34", "md5": "785f714f65b51188a67fdab20c14a664", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}]