EXPERIMENT_TITLE: GSM4873116: WT-HUDEP2 cell 2 capture HiC; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Approximately 2 million cells of each HUDEP-2 clone were differentiated for 5 days and fixed in 1% formaldehyde. Hi-C libraries were generated using the Arima-HiC kit, according to the manufacturer's protocols. Final amplification cycle numbers for each library were determined by qPCR in the QC2 step of the Arima protocol. Quantification of the libraries was performed with KAPA Library Quantification Kit (Roche, KK4824). For capture HiC 150-500 ng of the previously created Hi-C libraries were diluted to 25 uL and mixed with 2.5 ug Cot-1 DNA and 10 ug salmon sperm DNA and heated to 95°C for 5 minutes, then held at 65°C for at least 5 minutes. 33 uL of prewarmed (to 65°C) hybridization buffer (10X SSPE, 10X Denhardt's buffer, 10mM EDTA, and 0.2% SDS), along with 6 uL of RNA probe mixture (500 ng RNA probes and 20U RNase inhibitor) were added to the DNA mixture and hybridized for 24 hr at 65°C. After the hybridization incubation was complete, 50 uL of Streptavidan T1 beads (Dynabeads, Life Technologies) were washed in Bind-and-Wash buffer (1M NaCl, 10mM Tris-HCl, pH 7.5, and 1mM EDTA), resuspended in 134 uL of the same buffer, and then added to the hybridization mixture. The beads were allowed to bind to the biotinylated, hybridized DNA and RNA mixture for 30 minutes at room temperature before separating and discarding the supernatant. The bead-bound DNA was then washed once with low-stringency wash buffer (1X SSC, 0.1% SDS), for 15 minutes at room temperature, and three times with high-stringency wash (0.1X SSC, 0.1% SDS) for 10 minutes at 65°C, separating the beads on a magnet each time before discarding the supernatant. After the last wash, the beads were resuspended in 21 uL nuclease-free water. 1 uL was diluted 1:1000 and had qPCR performed as for QC2 of the Arima protocol to determine the number of cycles to amplify the enriched libraries. The indexes were already attached during Hi-C library prep, and remain the same. HiC and Capture HiC
STUDY_DESCRIPTOR_STUDY_ABSTRACT: We have engineered the 3 prime HS1 CTCF binding site at the human beta globin gene locus in HUDEP-2 cells and performed RNA-seq/ChIP-seq/ATAC-seq as well as HiC and capture HiC to dissect its potential in regulating gene expression Overall design: We have deleted 3 prime HS1 CTCF binding site in HUDEP-2 cells with CRISPR/Cas9 and inverted 3 prime HS1 CTCF binding site with CRISPR/Cas9-mediated HDR. In each condition, we have obtained two independent clones for the 3'HS1 CTCF binding site deletion and the 3 prime HS1 CTCF binding site inversion.
SAMPLE_TITLE: WT-HUDEP2 cell 2 capture HiC
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Erythroid progenitor"}, {"TAG": "cell type", "VALUE": "erythroid"}]
Pool_Member_@attributes_sample_title: WT-HUDEP2 cell 2 capture HiC
RUN_SET_RUN_Pool_Member_@attributes_sample_title: WT-HUDEP2 cell 2 capture HiC
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": null, "@attributes": {"cluster": "public", "filename": "WT-HUDEP2-CHiC_R1_001.fastq.gz", "size": "4020074674", "date": "2020-10-29 14:20:41", "md5": "18374b33d0665277809c72bb6754cf9c", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "WT-HUDEP2-CHiC_R2_001.fastq.gz", "size": "5307870009", "date": "2020-10-29 14:22:04", "md5": "c19b9ad568f452d8ee8dff544454fe72", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR12935508", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR12935508/SRR12935508", "size": "7705243318", "date": "2020-10-29 14:24:57", "md5": "0a2b1ac3d5c0435616e931ee29a6deeb", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR12935508.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos3/sra-pub-zq-22/SRR012/12935/SRR12935508/SRR12935508.lite.1", "size": "4662977234", "date": "2022-07-21 00:08:03", "md5": "3d20040f03e90120e7ca669a6767ddbe", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}]