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| Status |
Public on Sep 29, 2022 |
| Title |
cHiC__ESC_G4_wt_mouse_Rep-1 |
| Sample type |
SRA |
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| Source name |
ESC
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| Organism |
Mus musculus |
| Characteristics |
group: G4
genotype: wt
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| Treatment protocol |
Disassociated ESCs and limb cells were transferred to a 50-ml falcon tube and complemented with 10% FCS/PBS. 37% formaldehyde was added to a final concentration of 2% and cells were fixed for 10 min at room temperature. Crosslinking was quenched by adding glycine (final concentration; 125 mM). Fixed cells were washed twice with cold PBS and lysed using fresh lysis buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA with protease inhibitor) to isolate nuclei. Cell lysis was assessed microscopically after 10-min incubation in ice. Nuclei were centrifuged for 5 min at 480g, washed once with PBS and snap frozen in liquid N2.
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| Growth protocol |
Mouse G4 ESCs (XY, 129S6/SvEvTac x C57BL/6Ncr F1 hybrid) were grown as described previously on a mitomycin-inactivated CD1 mouse embryonic fibroblast feeder monolayer on gelatinised dishes at 37oC, 7.5% CO2 (Andrey and Spielmann, 2017; George et al., 2007). ESCs were cultured in ESC medium containing knockout DMEM with 4,5 mg/ml glucose and sodium pyruvate supplemented with 15% FCS, 10 mM Glutamine, 1x penicillin/streptomycin, 1x non-essential amino acids, 1x nucleosides, 0.1 mM beta-Mercaptoethanol and 1000 U/ml LIF. Medium was changed every day while G4-cells were split every 2-3 days or were frozen at 1x 106 cells/cryovial in ESC medium containing 20% FCS and 10% DMSO. ESCs and feeder cells were tested for Mycoplasma contamination using the MycoAlert detection kit and MycoAlert Assay Control Set. E11.5 forelimb cells were isolated from C57BL/6 embryonic limbs through trypsinization, filtration (100 µm) and centrifugation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
3C libraries were prepared from fixed nuclei as described previously (Kragesteen et al, 2018). Briefly, lysis buffer was removed by centrifugation at 400 g for 5 min at 4 °C, followed by supernatant aspiration, snap-freezing, and pellet storage at − 80 °C. Later, nuclei pellets were thawed on ice, resuspended in 520 μl 1× DpnII buffer, and then incubated with 7.4 μl 20% SDS shaking at 900 rpm. at 37 °C for 1 h. Next, 75 μl 20% Triton X-100 was added and the pellet was left shaking at 900 rpm at 37°C for 1 h. A 15-μl aliquot was taken as a control for undigested chromatin (stored at − 20°C). The chromatin was digested using 40 μl 10 U/μl DpnII buffer shaking at 900 rpm at 37°C for 6 h; 40 μl of DpnII was added and samples were incubated overnight, shaking at 900 rpm. at 37°C. On day three, 20 μl DpnII buffer was added to the samples followed by shaking for an additional 5 h at 900 rpm. at 37 °C. DpnII subsequently was inactivated at 65 °C for 25 min and a 50-μl aliquot was taken to test digestion efficiency (stored at − 20 °C). Next, digested chromatin was diluted in 5.1 ml H2O, 700 μl 10× ligation buffer, 5 μl 30 U/μl T4 DNA ligase and incubated at 16°C for 4 h while rotating. Ligated samples were incubated for a further 30 min at room temperature. Chimeric chromatin products and test aliquots were de-cross-linked overnight by adding 30 μl and 5 μl proteinase K, respectively, and incubated at 65 °C overnight. On the fourth day, 30 μl or 5 μl of 10 mg ml−1 RNase was added to the samples and aliquots, respectively, and incubated for 45 min at 37°C. Next, chromatin was precipitated by adding 1 volume phenol-chloroform to the samples and aliquots, vigorously shaking them, followed by centrifugation at 4,000 rpm at room temperature for 15 min. To precipitate aliquot chromatin, 1 volume 100% ethanol and 0.1 volume 3M NaAc, pH 5.6 was added and the aliquots placed at -80°C for 30 min. DNA was then precipitated by centrifugation at 5,000 rpm. for 45 min at 4°C followed by washing with 70% ethanol, and resuspension in 20 μl with 10 mM Tris-HCl, pH 7.5. To precipitate samples, extracted sample aqueous phases were mixed with 7 ml H2O, 1 ml 3M NaAc, pH 5.6, and 35 ml 100% ethanol. Following incubation at −20°C for at least 3 h, precipitated chromatin was isolated by centrifugation at 5,000 rpm for 45 min at 4 °C. The chromatin pellet was washed with 70% ethanol and further centrifuged at 5,000 rpm for 15 min at 4 °C. Finally, 3C library chromatin pellets were dried at room temperature and resuspended in 10 mM Tris-HCl, pH 7.5. To check the 3C library, 600 ng were loaded on a 1% gel together with the undigested and digested aliquots.
The 3C library was then sheared using a Covaris sonicator (duty cycle: 10%; intensity: 5; cycles per burst: 200; time: 6 cycles of 60 s each; set mode: frequency sweeping; temperature: 4–7 °C). Adaptors were added to the sheared DNA and amplified according to the manufacturer’s instructions for Illumina sequencing (Agilent). The library was hybridised to the custom designed SureSelect beads and indexed for sequencing (75–100 bp paired-end) following the manufacturer’s instructions (Agilent).
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| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
mpimg_L13065_L13736_ESC-G4-cHiC-_S2_S6_merged_enriched_only_MAPQ30.hic
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| Data processing |
Raw fastq files had read lengths of 75bp and 100bp, respectively. In a preprocessing step, fastq files with 100 bp read length were trimmed to 75bp to achieve comparable initial read lengths for all samples.
Fastq files were processed with the HiCUP pipeline v0.8.1 (no size selection, Nofill: 1, Format: Sanger) for mapping, filtering and deduplication steps (Wingett et al., 2015). The pipeline was set up with Bowtie 2.4.2 for mapping short reads to reference genome mm10 (Langmead and Salzberg, 2012). If replicates were available, they were merged after the processing with the HiCUP pipeline. Binned and KR normalized cHiC maps (Knight and Ruiz, 2012; Rao et al., 2014) were generated using Juicer tools v1.19.02 (Durand et al., 2016). Only read pairs for region chr8:39,030,001-48,000,000 and with MAPQ≥30 were considered for the generation of cHiC maps.
Additional to the original cHiC maps, custom reference genomes were derived from mm10 for the three deletions (ΔD1, ΔD2, ΔD1+2), considering the respective deletions and cHiC data was processed correspondingly. cHiC maps were displayed as heatmaps in which very high values were truncated to improve the visualization.
Genome_build: mm10 and custom mutant genomes
Supplementary_files_format_and_content: .hic .mutants are mapped on both wt and corresponding mutant mm10 genomes (see description for each sample)
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| Submission date |
Oct 13, 2021 |
| Last update date |
Sep 29, 2022 |
| Contact name |
Michael I Robson |
| E-mail(s) |
robson@molgen.mpg.de
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| Organization name |
Max Planck Institute for Molecular Genetics
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| Street address |
Max Planck Institut für molekulare Genet, Ihnestrasse 63-73
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| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE185775 |
Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes |
| GSE185847 |
Promoter repression and 3D-restructuring resolves divergent developmental gene expression in TADs [cHiC] |
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| Relations |
| BioSample |
SAMN22308124 |
| SRA |
SRX12623948 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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