EXPERIMENT_TITLE: GSM4624641: Hi-C-Clone21-CTCF&TSS-CRISPR#1-rep2-1st-seq; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: In situ Hi-C was performed as previously described (Rao et al., Cell, 2014; Nora et al., Cell, 2017; Huang et al., Genes Dev. 2017.). Briefly, ~10 million HAP1 cells, WT, Clone 21, Clone 25, and subsequent CRISPR-edited clones were crosslinked in 2% formaldehyde at room temperature for 10 minutes, and then quenched in 0.125M glycine for 5 minutes, on an orbital shaker. Cells were then transferred from a culture flask to a 15mL tube, pelleted, washed with 1mL cold PBS, transferred to an Eppendorf tube, pelleted, resuspended in 1mL cold Cell Lysis Buffer (10mM Tris pH 8.0, 10mM NaCl, and 0.2% NP-40/Igepal), and incubated on ice for 10 minutes. Nuclei were then pelleted, washed once using 800µL NEBuffer™ DpnII, pelleted, and resuspended in 500µL NEBuffer™ DpnII. A final concentration of 0.3% SDS was added, and samples were incubated at 37°C, 950 rpm for 1 hour in a thermomixer. A final concentration of 1.8% Triton X-100 was then added to each sample, which was subsequently incubated at 37°C, 950 rpm for 1 hour. 40µL nuclease-free water, as well as 300U DpnII (NEB R0543M), was added, and samples were digested at 37°C, at 950 rpm overnight in a thermomixer. Another 300U DpnII were added for an additional 4 hours of digestion with mixing. The samples were then incubated at 65°C, 950 rpm for 20 minutes. Nuclei were then pelleted, resuspended in 1X NEBuffer™ 2, with biotin-14-dATP, dTTP, dCTP and dGTP and DNA polymerase I, Large (Klenow) Fragment (NEB M0210), and incubated at 37°C, 950 rpm in a thermomixer for 90 minutes. Ligation reaction was subsequently carried out in a total volume of 1.2mL with 4000U T4 DNA Ligase (NEB) at 16°C for 4 hours, followed by 30 minutes at room temperature. 20µL of 20mg/mL Proteinase K and 120µL of 10% SDS were then added to each sample, followed by crosslinking reversal at 65°C overnight. An additional 10µL proteinase K was added to each sample, which was incubated at 55°C for 2 hours. 2µL DNase-free RNAase was added to each sample, followed by incubation at 37°C for 30 minutes. Phenol-chloroform extraction was performed to purify DNA. DNA was sonicated to 200-300bp in a bath sonicator (QSonica Q800R3), followed by beads clean-up. Biotinylated nucleotides-filled ligation junctions were pulled down with 50µL Dynabeads™ MyOne™ Streptavidin C1 (Thermo Fisher 65001). Sequencing libraries were subsequently prepared: End Repair, dA-Tailing, adaptor ligation, followed by 6 cycles of indexing PCR. The quality and size (average 429 bp) of each library was evaluated using the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA), followed by quantitation using real-time PCR using the KAPA Library Quant Kit for Illumina (KAPA Biosystems, KK4835). Libraries were then pooled and sequenced in paired-end mode on the NextSeq 500 to generate 2 x 75 bp reads using Illumina-supplied kits as appropriate.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Mammalian chromosomes are partitioned into contact domains that can be conserved as functional units in evolution. Disruptions of domains can result from perturbed CTCF, cohesin, or chromosomal rearrangements. However, to what extent domains can be created de novo has not been explored in depth. Here, using a gain-of-function approach leveraging genome editing and Hi-C, we examined whether, and how, a putative boundary element can function to organize de novo domains in the context of multiple ectopic insertion sites. We subsequently dissected the distinct roles of the CTCF binding site and the transcription start site within the insertion element in changing genome folding. Overall design: Using a transposon-based approach, we inserted a 2 kb DNA fragment, which resides within a tissue-invariant domain boundary, into the genomes of near-haploid HAP1 cells. Following transposition, we established clonal lines and prioritized Clones 21 (C21) and 25 (C25), which contained ten and six insertions, respectively, across a total of 11 chromosomes. We then performed in situ Hi-C on edited clones and unedited parental (WT) cells to characterize how the insertion of a boundary-associated DNA fragment shapes human genome architecture. Furthermore, we carried out a series of CRISPR-based editing experiments to dissect how the TSS and the CTCF binding site within the insertion element contributes to new domain formation. Additionally, we used CRISPR to delete the endogenous 2 kb element and characterized its function at its endogenous boundary. In addition to Hi-C, we performed Capture-C, ChIP-seq (CTCF, Rad21, and H3K27Ac), and RNA-seq to further characterize the structural changes resulting from each round of genome editing experiments.
SAMPLE_TITLE: Hi-C-Clone21-CTCF&TSS-CRISPR#1-rep2-1st-seq
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HAP1"}, {"TAG": "cell type", "VALUE": "KBM7-derived near haploid cells"}]
Pool_Member_@attributes_sample_title: Hi-C-Clone21-CTCF&TSS-CRISPR#1-rep2-1st-seq
RUN_SET_RUN_Pool_Member_@attributes_sample_title: Hi-C-Clone21-CTCF&TSS-CRISPR#1-rep2-1st-seq
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