EXPERIMENT_TITLE: GSM6203883: Ki67_aid_HCT116_IAA_interphase_HiC_r2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: 1. pA-DamID: 1 million permeabilized cells were processed with pA-DamID (van Schaik, 2020) with a primary antibody and for a Dam control condition, to specifically deposit m6A marks at chromatin nearby the primary antibody. Genomic DNA was isolated using the Bioline Isolate II Genomic DNA Kit; 2. RNA-seq: RNA was isolated using the Qiagen RNeasy column purification kit; 3. Repli-Seq: cells were harvested as for pA-DamID and immediately fixed in ice-cold ethanol. 4. ChIP-seq: Cells were crosslinked (1% FA, 10 minutes) and quenced with glycine (2.0M). 5. Hi-C: Cells were harvested and crosslinked (2% FA, 10 minutes). 1. pA-DamID: the full protocol is described in Leemans, 2019, except that the DpnII digestion is omitted. 500 ng of genomic DNA was digested at m6A-methylated GATC sequences with DpnI and ligated to adapters. DNA fragments were amplified and further processed with end-repair, 3A-overhang tailing and Y-shaped adapter ligation, before a final round of 10-12 PCR cycles with illumina index adapters; 2: RNA-seq: sequencing libraries were prepared using the Illumina TruSeq polyA stranded RNA kit; 3. Repli-seq: replicated DNA was enriched for with a BrdU IP, before libraries were prepared as described in Marchal, 2018; 4. ChIP-seq: Chromatin was fragmented to ~300 bp fragments with a Bioruptor Plus sonication device (Diagenode). Ki-67 antibody (ab15580, Abcam; 5 μL per ChIP) was coupled with Protein G beads (Thermo Fisher Scientific) and incubated with fragmented chromatin overnight at 4 °C. Released DNA fragments were purified with the MinElute PCR Purification kit (Qiagen). The purified DNA fragments were prepared with the KAPA HTP Library Preparation kit (Roche); 5. Hi-C: Cross-linked nuclei were digested with MboI and biotinylated nucleotides incorporated in the overhangs. Following blunt-end ligation, the ligated fragments were extracted using streptavidin pulldown and prepared for sequencing using a standard end-repair / 3A-tailing procedure.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Ki67 is a widely-used proliferation marker with functions in chromosomal organization. It forms a protective layer around mitotic chromosomes and is involved in genome positioning in interphase. However, a detailed understanding of how Ki67 organizes the genome was still lacking, partially because genome-wide interaction profiles were missing. Here, we generate DNA-Ki67 interaction profiles using our pA-DamID method, and show that Ki67 interacts with the genome in a domain-like pattern that partially overlaps with lamina-associated domains. Overall design: DNA-Ki67 interactions were profiled with pA-DamID in three human cell lines, throughout the cell cycle and following various nucleolar perturbation experiments. Additionally, Ki67 interactions were compared to Lamin B1 interactions and H3K27me3 and H3K9me3 in normal and perturbed conditions. Finally, the functional role of Ki67 was determined by profiling gene expression data (RNA-seq), replication timing (Repli-seq) and genome interactions (Hi-C).
SAMPLE_TITLE: Ki67_aid_HCT116_IAA_interphase_HiC_r2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HCT116 (ATCC CCL-247)"}, {"TAG": "cell type", "VALUE": "Colorectal carcinoma cells"}, {"TAG": "library type", "VALUE": "Hi-C"}, {"TAG": "genetic modification", "VALUE": "OsTir1; Ki67-miniAID-mClover"}, {"TAG": "experiment", "VALUE": "Ki67_aid"}, {"TAG": "condition", "VALUE": "interphase_IAA"}]
Pool_Member_@attributes_sample_title: Ki67_aid_HCT116_IAA_interphase_HiC_r2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: Ki67_aid_HCT116_IAA_interphase_HiC_r2
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