EXPERIMENT_TITLE: GSM4798673: NPC Hi-C_rep1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: For ATAC-seq, 50000 cells were washed with cold-PBS and resuspended by cell lysis buffer (10mM Tris-HCl (pH 7.4), 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630) and centrifuged (10min, 2000g at 4°C). For H3K27ac ChIP-seq, CCells were washed with DPBS and crosslinked by crosslinking buffer (100mM NaCl, 0.1mM EDTA, 5mM HEPES (pH8.0), 1% formaldehyde) for 10min at room temperature. The crosslinking process was halted with 125mM glycine in room temperature for 5 min with rotation and washed twice with cold DPBS. Crosslinked samples were resuspended in SDS lysis buffer (1%SDS, 50mM Tris-HCl (pH8.0), 10mM EDTA) with protease inhibitor. For Hi-C, about 1 million cells were used for crosslinking with 1% formaldehyde. Crosslinked cells were lysed (10nM Tris-HCl (pH8.0), 10mM NaCl, and 0.2% IGEPAL CA630) and digested with 100U MboI. For ATAC-seq, the nuclei pellet were resuspended with Transposase mixture in Illumina Nextera Kit (FC-121-1031) and incubated for 30min at 37°C. Tagmented DNA fragments were puriﬁed using MinElute PCR purification kit following manufacturer's protocol. For eluted DNA samples, we executed small scale qPCR to determine adequate PCR amplification cycles for library preparation. We checked Ct value for qPCR sample mix (5ul 2×KAPA HiFi HotStart Ready Mix, 0.1ul 100x SYBR green, 0.3ul Nextera Ad1.2 (25uM), 0.3ul Nextera Ad2.X (25uM), 2.3ul Nuclease-free water and 2ul eluted DNA) and determined PCR amplification cycles with value of Ct+1. We then executed large scale PCR (25ul 2×KAPA HiFi HotStart Ready Mix, 1.5ul Nextera Ad1.2 (25uM), 1.5ul Nextera Ad2.X (25uM), 2ul Nuclease-free water and 20ul eluted DNA). After PCR step, we cleaned library samples and performed size selection (200~400bp) by AMPure XP beads (Beckman Coulter, A63881) following manufacturer's instruction. For H3K27ac ChIP-seq, sonicated chromatin samples were incubated with H3K27ac antibody and magnetic bead for 4 hours in 4°C with rotation. At the same time, input chromatin was stored from sonicated samples to be used as input control. After incubation, the bead complex was washed by RIPA-α (140mM NaCl, 1mM EDTA pH8, 0.5mM EGTA pH8, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 10mM Tris pH8) with 4 times. The chromatin immunoprecipitated complex was treated with RNaseA and executed overnight reverse-crosslinking at 68°C. The chromatin immunoprecipitated DNA was extracted using AMPure XP beads and libraries were prepared using NEB Next Ultra II DNA library Prep Kit. For Hi-C library, digested fragments were labelled with biotin-14-dCTP and proximally ligated with T4 DNA Ligase, followed by reverse-crosslinking. The fragments were then subjected to sonication and purified with AMPure XP beads. The final ligated DNA was pulled down with Dynabeads MyOne streptavidin T1 beads.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: We generated ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) data and Hi-C data for induced pluripotent stem cell (iPSC), neuronal progentior cell (NPC), and investigated cell type-specific open chromatin regions and related enhancers Overall design: ATAC-seq for iPSC and NPC. Hi-C for iPSC and NPC. H3K27ac ChIP-seq for iPSC and NPC.
SAMPLE_TITLE: NPC Hi-C_rep1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "induced pluripotent stem cell"}, {"TAG": "cell type", "VALUE": "Human induced pluripotent stem cell derived"}, {"TAG": "developmental stage", "VALUE": "Neuronal progenitor cell"}]
Pool_Member_@attributes_sample_title: NPC Hi-C_rep1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: NPC Hi-C_rep1
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": null, "@attributes": {"cluster": "public", "filename": "3-NPC-rep1-Hic_1.fastq.gz", "size": "14894744490", "date": "2020-09-22 19:18:31", "md5": "c50f21c03e90f2f5fa3e89fe494a1552", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "3-NPC-rep1-Hic_2.fastq.gz", "size": "19524241264", "date": "2020-09-22 19:22:25", "md5": "386a55d726398c9a281c37105686ceaf", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR12694370", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR12694370/SRR12694370", "size": "23730906897", "date": "2020-09-22 19:27:23", "md5": "e045e1b4074d857b0f1e5fba5e3477d6", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR12694370.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos3/sra-pub-zq-22/SRR012/12694/SRR12694370/SRR12694370.lite.1", "size": "14140537121", "date": "2022-07-15 04:08:17", "md5": "5859e9bb26791911106eb3103fae0de9", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}]