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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 27, 2022 |
| Title |
HiC of Nng2 overexpressing cells rep3 |
| Sample type |
SRA |
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| Source name |
Electroporated cells E15.5 somatosensory cortex
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: E15.5
celltype: GFP+
technique: HiC
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The somatosensory cortex of in utero electroporated E14.5 mouse embryos was isolated and the electroporated area dissected. Single cell suspension was prepared using the papain-based neural dissociation kit (Miltenyi Biotec, Cat. N: 130-092-628) according to the manufacturer protocol with reduced dissociation times (5 min. instead of 15 min. at 37˚C). Cell number as well as the viability was assessed using the Countess™ II Automated Cell Counter (Invitrogen). Dissociated cells were fixed for 10 min. in 1% Formaldehyde (ThermoFisher, Cat. N: 28908), quenched by addition of Glycine (Invitrogen, Cat. N: 15527013) to a final concentration of 0.2M, washed in PBS with 0.1% BSA, passed through a 40µM cell strainer (ThermoFisher, Cat. N: 15342931) and immediately FAC-sorted at a BD FACSAria Fusion (BD Bioscience) with four lasers (405, 488, 561, 640) using a 100µm nozzle. Sorted cells were lysed with 0.2% Igepal-CA630 (Sigma-Aldrich, Cat. N: I3021), permeabilized with 0.5% SDS (Invitrogen, Cat. N:AM9823) and digested with 200U DpnII (New England Biolabs, Cat. N: R0543) overnight at 37˚C. Subsequently, sticky ends were filled by incubating the nuclei for 4h at RT with DNA Polymerase I (New England Biolabs, Cat. N: M0210) and a biotin-14-dATP (Life Technologies, Cat. N: 195245016) containing nucleotide mix in DpnII buffer followed by proximity ligation for at least 6h at 16°C. Thereafter nuclei were lysed, proximity ligated DNA was reverse-crosslinked overnight at 68˚C followed by a purification by ethanol precipitation and shearing to ~550 bp DNA fragments using a Covaris S220 sonicator.
Biotin pulldown was performed by incubation the sheared DNA with MyOne Streptavidin T1 beads (ThermoFisher, Cat. N: 65602) for 30min. at RT followed by on-bead biotin removal and end-repair by incubating the samples for 30 min. at RT in a reacting mix consisting of T4 DNA Polymerase, T4 Polynucleotide Kinase and DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs, Cat. N: M0203, M0201, M0210). Thereafter A-tailing using Klenow Fragment exo-minus (New England Biolabs, Cat.N.: M0212) was performed for 30 min. at 37˚C followed by ligation of NextFlex DNA barcodes (Perkin Elmer, Cat. N.: NOVA-514101) using the NEB Quick Ligase (New England Biolabs, Cat. N: M2200). Between each of the incubation steps the samples bound to the streptavidin beads were washed twice with washing buffer containing 0.05% Tween-20 (Sigma-Aldrich, Cat. N: P9416) and once with the respective buffer of the following reaction. Libraries were amplified on the streptavidin beads using the NEBNext Ultra Q5 II Master mix (New England Biolabs, Cat. N.: M0544) using following program: 98˚C 30s; {98˚C 10s, 65˚C 75s} x10; 65˚C 5min; Hold at 10˚C. After the amplification the streptavidin beads were pelleted and the supernatant was purified twice using 0.7x AMPure XP beads (Agencourt, Cat. N: A63881) to reach an average fragment size of approximately 500bp.
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| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
processed data: Compressed text file, containing pair-wise contact infomration in the medium juicer format (https://github.com/aidenlab/juicer/wiki/Pre) but omitting the read name: <str1> <chr1> <pos1> <frag1> <str2> <chr2> <pos2> <frag2> <mapq1> <mapq2>.
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| Data processing |
Hi-C data were mapped to the mm10 genome using Juicer (Rao et al., 2014). Only uniquely mapping reads (mapq>30) were retained for further analysis. After removal of PCR duplicates, reads were translated into a pair of fragment-ends (fends) by associating each read with its downstream fend.
Genome_build: mm10
Supplementary_files_format_and_content: Compressed text file, containing pair-wise contact infomration in the medium juicer format (https://github.com/aidenlab/juicer/wiki/Pre) but omitting the read name: <str1> <chr1> <pos1> <frag1> <str2> <chr2> <pos2> <frag2> <mapq1> <mapq2>.
Supplementary_files_format_and_content: Bigwig files with CpG or GpC methylation levels of all cytosines covered by at last 5 reads (5x coverage).
Supplementary_files_format_and_content: Tab seperated table containing read counts per gene.
Supplementary_files_format_and_content: Compressed files contain sequences of selecred CREs (design.fa.gz), their annotation (labels.tsv.gz) and a pickle library with filtered CRE - Barcode associations (CRE_BC_association.pickle.gz).
Supplementary_files_format_and_content: Compressed tarball archive containing DNA/RNA read counts and annotations for both replicates.
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| Submission date |
Sep 16, 2021 |
| Last update date |
Jan 27, 2022 |
| Contact name |
Boyan Bonev |
| E-mail(s) |
[email protected]
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| Organization name |
Helmholtz Zentrum München
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| Lab |
Bonev Lab
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| Street address |
Ingolstädter Landstr. 1
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| City |
Neuherberg |
| State/province |
Deutschland |
| ZIP/Postal code |
85764 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE155677 |
Multimodal profiling of the transcriptional regulatory landscape of the developing mouse cortex identifies Neurog2 as a key epigenome remodeler |
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| Relations |
| BioSample |
SAMN21463012 |
| SRA |
SRX12214119 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5583714_NGN2_HiC_IUE24h_rep3.txt.gz |
4.1 Gb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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