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| Status |
Public on Jan 11, 2017 |
| Title |
Kc167_Hi-C_primary |
| Sample type |
SRA |
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| Source name |
Kc167 cultured cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
developmental stage: embryonic
cell line: Kc167
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| Treatment protocol |
1 billion Kc167 cells at a density of 4-6 x 10^6 cells/mL were washed with 20 mL CMM3 media and then cross-linked with 1% EM-grade paraformaldehyde.
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| Growth protocol |
Kc167 cells were obtained from the Drosophila Genomics Resource Center (Bloomington, IN). Cells were grown in CCM3 media (HyClone) at 25° C following Drosophila Genomics Resource Center protocols.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cross-linked chromatin was released by detergent lysis and Dounce homogenization. Cross-linked proteins were then biotinylated at cysteine residues and the DNA digested with DpnII. Digested chromatin was bound to streptavidin beads, thoroughly washed to remove uncross-linked DNA, DNA ends filled in with biotin-14-dATP, and free DNA ends ligated together. DNA-protein cross-links were reversed, DNA purified, and biotinylated nucleotides marking unligated ends removed.
DNA sheared to a size of approximately 500 bp. DNA was end-repaired and adaptor-ligated by following “NEBNext End Prep” and “Adaptor Ligation” in the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). The biotinylated DNA was pulled down with streptavidin beads and PCR amplified by following "PCR Amplification" in the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Platforms: Illumina NextSeq 500 (used to generate majority of raw files), HiSeq X Ten
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| Data processing |
Reads were mapped to dm3/BDGP Release 5 of the Drosophila melanogaster genome using BWA-MEM.
Reads were assigned to DpnII restriction fragments, duplicates removed, reads with a MAPQ < 30 removed, and intra-fragment reads removed.
Genome was then divided into equally spaced bins.
To account for the uncertainty in the location of the position of the cross-link within each restriction fragment,the read position was radnomized within the restriction fragment each read mapped to and the resulting contact was assigned to its respective genomic bin.
Hi-C contact maps were normalized by matrix balancing using the KR normalization algorithm (Durand et al., 2016; Knight and Ruiz, 2012; Rao et al., 2014).
Biological replicates combined after filtering, intrafragment read positions randomized as described above, and the combined contact map KR normalized. All subsequent analysis was performed on the combined, KR normalized contact map.
TADs were identified using the previously described Arrowhead algorithm (Durand et al., 2016; Rao et al., 2014), with an additional post-processing step: TADs were identified at 5 kb, 2 kb, 1 kb, and 500 bp resolution using the Arrowhead algorithm, merged into a single list sorted by decreasing corner score, and conflicts, defined as the boundary of one TAD being located within another TAD, resolved by using the greater corner score of any conflicting TADs.
Loops were annotated by visual identification of focal peaks of contact enrichment using Juicebox (Durand et al., 2016).
All of the above steps were performed using the previously described Juicer pipeline (Durand et al., 2016; Rao et al., 2014) and Juicebox visualization interface (Durand et al., 2016).
Genome_build: dm3
Supplementary_files_format_and_content: *.hic file of Hi-C contacts in compressed format. Readable by open-source Juicebox software (http://aidenlab.org/juicebox/).
Supplementary_files_format_and_content: *_randomized.hic file of Hi-C contacts in compressed format after read positions were randomized within the restriction fragment to which each read was assigned. Readable by open-source Juicebox software (http://aidenlab.org/juicebox/).
Supplementary_files_format_and_content: Kc167combined_TADs.txt file of TAD calls from combined biological replicates in Juicebox format. The column headers x1 x2 y1 y2 represent the boundaries of the TAD in genomic coordinates.
Supplementary_files_format_and_content: Kc167combined_loops.txt file of loop calls from combined biological replicates in Juicebox format. The column headers x1 x2 represent the position of the loop anchor closer to the start of the chromosome; y1 y2 represent the position of the loop anchor closer to the end of the chromosome.
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| Submission date |
Oct 24, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Kyle Eagen |
| E-mail(s) |
[email protected]
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| Organization name |
Baylor College of Medicine
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| Department |
Department of Molecular and Cellular Biology
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| Lab |
Eagen Lab
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| Street address |
1 Baylor Plaza
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL19132 |
| Series (1) |
| GSE89112 |
Sub-kilobase resolution Kc167 cell Hi-C |
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| Relations |
| BioSample |
SAMN05936438 |
| SRA |
SRX2266515 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2358971_Kc167primary.hic |
1.1 Gb |
(ftp)(http) |
HIC |
| GSM2358971_Kc167primary_randomized.hic |
1.1 Gb |
(ftp)(http) |
HIC |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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