EXPERIMENT_TITLE: GSM6922384: RPE TP53-/-, control, rep1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: 1-2 million cells were collected and fixed with 2% formaldehyde (Thermo Fisher Scientific, catalogue no. 11483217). The reaction was quenched with 200mM glycine (VWR, catalogue no. 101194M) final concentration, cells were washed with PBS (Thermo Fisher Scientific, catalogue no. 10010023) and lysed in a solution containing 10mM Tris-HCl pH 8.0 (Thermo Fisher Scientific, catalogue no. 15568025), 10mM NaCl (Sigma-Aldrich, catalogue no. S6546), 0.2% Igepal CA-630 (Sigma-Aldich, catalogue no. I8896), and 1x Proteinase inhibitor cocktail (Roche, catalogue no. 11697498001) at 4°C for 30 minutes. Resulting nuclei were re-suspended in 1X NEB3.1 buffer (New England Biolabs, catalogue no. B7203S). Nuclei suspension was incubated with 0.11% SDS (Carl Roth, catalogue no. CN30) final concentration for 10 minutes at 65°C. Reaction was quenched with 1% Triton-X100 (AppliChem, catalogue no. A1388) and nuclei were digested with 100U MboI restriction enzyme (New England Biolabs, catalogue no. R0147) at 37°C overnight. Restriction enzyme was inactivated according to manufacturer's specifications and digested nuclei were washed and re-suspended in 1x NEB3.1. Digested ends were then marked with biotin via incubation in 0.03mM biotin-14-dATP (Thermo Fisher Scientific, catalogue no. 19524016), 0.03mM dCTP, 0.03mM dGTP, 0.03mM dTTP (Promega, catalogue no. U1420) and 50U Klenow DNA polymerase I (New England Biolabs, catalogue no. M0210M) for 4 hours at room temperature. Resulting blunt-ends were proximally ligated with 50U T4 DNA Ligase (Thermo Fisher Scientific, catalogue no. EL0011), 1x T4 DNA ligase buffer (Thermo Fisher Scientific, catalogue no. B69), 5% PEG, 1% Triton-X100, 0.1mg/ml BSA (New England Biolabs, catalogue no. B9000S) for 4 hours at room temperature. Crosslink reversal was performed on the proximity-ligated chromatin through the incubation with 300mM NaCl and 1% SDS overnight, at 68°C. The sample was then treated with 50g/ml RNase A (Thermo Fisher Scientific, catalogue no. EN0531) for 30 minutes at 37°C, followed by 400ug/ml Proteinase K (Promega, catalogue no. V3021) at 65°C for one hour. DNA was purified via precipitation with 1.6 volumes of pure ethanol and 0.1 volumes of sodium acetate, pH 5.2 (Thermo Fisher Scientific, catalogue no. R1181) at -80°C. DNA was eluted and then fragmented by sonication at 80V peak incidence power, 10% duty factor, 200 cycles per burst, for 60-80 seconds with an E220 Focused-ultrasonicator (Covaris). Sheared DNA was size selected for library preparation using AMPure XP beads (Beckman Coulter, catalogue no. A63881). Biotin-marked fragments were isolated with Dynabeads™ MyOne™ Streptavidin C1 (Thermo Fisher Scientific, catalogue no. 65001) and all subsequent steps were performed on the bead-bound DNA fraction. Hi-C library preparation continued with end polishing reaction, which involved incubation of DNA with 1x T4 ligase buffer (New England Biolabs, catalogue no. B0202S), 2.5mM each dNTP, 50U T4 Polynucleotide Kinase (New England Biolabs, catalogue no. M0201), 12U T4 DNA Polymerase (New England Biolabs, catalogue no. M0203), and 5U Klenow DNA polymerase I, at room temperature for 30 minutes. PolyA tail was added by incubating the DNA sample in 1X NEBuffer™ 2 (New Enlgand Biolabs, catalogue no. B7002S) with 0.5mM dATP and 25U Klenow Fragment (3'→5' exonuclease) (New England Biolabs, catalogue no. M0212) at 37°C for 30 minutes. DNA fragment ends were then ligated to Illumina TruSeq unique dual indexes (Integrated DNA Technologies) in 1X T4 Ligation buffer with 5% PEG and 15U T4 DNA Ligase for 2 hours at room temperature or overnight at 16°C. Lastly, libraries were PCR amplified using Illumina forward (AATGATACGGCGACCACCGAGATCTACAC) and reverse (CAAGCAGAAGACGGCATACGAGAT) primers and KAPA HiFi HotStart ReadyMix (Roche, catalogue no. KK2602) for 6-10 cycles. Resulting fragments were size-selected with AMPure XP beads.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Whole genome doubling (WGD) is a recurrent event in human cancers and it promotes chromosomal instability and acquisition of aneuploidies. However, the 3D organization of the chromatin in WGD cells and its contribution to oncogenic phenotypes are currently unknown. Here, we show that in p53 deficient cells WGD induces loss of chromatin segregation (LCS), characterized by reduced segregation between short and long chromosomes, A and B sub-compartments, and adjacent chromatin domains. LCS is driven by downregulation of CTCF and H3K9me3 in cells that bypassed activation of the tetraploid checkpoint. Longitudinal analyses revealed that LCS primed genomic regions for sub-compartment repositioning in WGD cells, which resulted in chromatin and epigenetic changes associated with oncogene activation in tumours ensuing from WGD cells. Importantly, sub-compartment repositioning events were largely independent of chromosomal alterations, indicating that these were complementary mechanisms contributing to tumour development and progression. Overall, LCS initiates chromatin conformation changes that ultimately result in oncogenic epigenetic and transcriptional modifications, suggesting that chromatin evolution is a hallmark of WGD-driven cancer. Overall design: For assessing chromatin organization immediately after WGD, chromatin conformation capture (Hi-C) was performed in pre- and post-WGD cells in RPE TP53-/-, CP-A TP53-/- (clone 3 and clone 19), and K562 cells. Distinct WGD inductions were used to exclude that the treatment itself and not WGD is responsible for changes observed at chromatin level. Additionally, cells exhibiting chromosomal instability only, but not WGD, (CIN-only) were considered to check whether CIN and WGD are leading to the same phenotype. Spontaneous high ploidy cells detected in the CP-A TP53-/- were sorted and assessed by Hi-C. Considering LCS was hypothesized to be caused by CTCF and H3K9me3 insufficiency due to defective tetraploidy checkpoint after WGD, Hi-C was performed in CP-A TP53-/- WGD cells were treated with CDK4/6 inhibitor to mimic the G1 extension assciated with a p53-induced tetraploidy checkpoint, as well as in CP-A p53 wild type WGD cells, since in both cases CTCF and H3K9me3 levels were rescued. Long-term post-WGD cells with tumorigenic properties were then assessed. Cells were grown both in vivo in NSG mice from RPE TP53-/- 20w pWGD cells (T1, T2, T3), and in vitro in soft agar as colonies, derived from CP-A TP53-/- clone 3 6w pWGD (C1, C2) and CP-A TP53-/- 20w pWGD (C1, C2). Each experiment was performed in replicates, as indicated. Samples derived from distinct WGD inductions are marked as "experiment 1/2" in the sample sheet. All conditions were compared to their respective controls.
SAMPLE_TITLE: RPE TP53-/-, control, rep1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "retinal epithelium"}, {"TAG": "tissue", "VALUE": "retinal epithelium"}, {"TAG": "cell line", "VALUE": "hTERT RPE-1"}, {"TAG": "cell type", "VALUE": "immortalized retinal epithelial cells"}, {"TAG": "genotype", "VALUE": "TP53-/-"}, {"TAG": "condition", "VALUE": "control"}]
Pool_Member_@attributes_sample_title: RPE TP53-/-, control, rep1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: RPE TP53-/-, control, rep1
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR23015982.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos4/sra-pub-zq-1/SRR023/23015/SRR23015982/SRR23015982.lite.1", "size": "11527307210", "date": "2023-01-12 17:44:58", "md5": "ebe431e180117e8daa2ea0260fd15384", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR23015982", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR23015982/SRR23015982", "size": "15680138402", "date": "2023-01-08 17:51:50", "md5": "61f36129f85384f822c5a57a5846ffbf", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "LambutaNanni2023_RPE_TP53_Ctrl_1_R2_HiC.fastq.gz", "size": "11705042633", "date": "2023-01-08 17:49:36", "md5": "a1b2531e1f77c30ecf2e427ac2b3e82a", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "LambutaNanni2023_RPE_TP53_Ctrl_1_R1_HiC.fastq.gz", "size": "11746166943", "date": "2023-01-08 17:49:01", "md5": "f38e12ad36e2a4430ef6fc108619b0bf", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]