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| Status |
Public on Feb 01, 2022 |
| Title |
knrl/kni - knrl tethers replacement1 |
| Sample type |
SRA |
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| Source name |
nc14 embryo
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| Organism |
Drosophila melanogaster |
| Characteristics |
developmental stage: nc14
tissue: embryo
genotype: AllGAF
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| Treatment protocol |
Embryos were collected in nylon mesh sieves, dechorionated for 3 mins in 3% sodium hypochlorite, rinsed with deionized water, and transferred to glass scintillation vials containing 6 mL PBST (0.1% Triton-X in PBS), 7.5 mL N-heptane, and 1.5 mL fresh 16% formaldehyde. Crosslinking was carried out at room temperature for exactly 15 mins on an orbital shaker at 250rpm, followed by addition of 5.5 mL 2M Tris-HCl pH7.5 and shaking for 5 mins to quench the reaction. Embryos were washed twice with >15mL PBST and stored at 4˚C while an additional 2-3 collection rounds were carried out (45 mins each). Pooled embryos were subjected to a secondary crosslinking in 15 mL DSG+EGS (ThermoFisher, 3 mM each) at room temperature for 45 mins on a rotating mixer. Following quenching by addition of 5.5 mL 2M Tris-HCl pH7.5 and rotation for 5 mins, the embryos were washed twice with PBST and stored at 4˚C overnight. Samples were hand-sorted the following day under a dissection microscope to remove inappropriately staged embryos, snap-frozen in liquid nitrogen and stored at -80˚C.
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| Growth protocol |
Embryos were collected on yeasted apple juice plates at 25˚C. Following two 1-hour pre-lays, embryos were collected for 45 mins and subsequently incubated at 25˚C for 2 hours to enrich for nc14 embryos.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was digested with a pre-determined amount of Micrococcal Nuclease (Worthington Biochem) to yield a 90% mononucleosome / 10% dinucleosome ratio given the appropriate number of embryos.
Micro-C libraries were constructed as previously described [Hsieh et al. 2020], with the following modifications.
Libraries were barcoded, pooled and subjected to paired-end sequencing on an Illumina Novaseq S1 100nt Flowcell (read length 50 bases per mate, 6-base index read).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Library strategy: Micro-C
Micro-C data was analyzed using the 4D Nucleome Hi-C analysis pipeline. Briefly, paired-end reads were mapped to the dm6 reference genome using BWA v0.7.17 [Li and Durbin, 2009], and alignments were filtered using pairtools v0.2.2 to retain only uniquely-mapping reads with alignment quality ≥3. Reads were assigned to 100bp genomic bins, and “inward”/”outward” reads assigned to adjacent bins (separated by less than 50bp) were removed. Matrix aggregation and normalization were performed using Cooler v0.8.3 [Abdennur and Mirny 2019], using the built-in ICE balancing method. Matrices were visualized using HiGlass [Kerpedjiev et al. 2018]. Visual inspection of contact matrices showed high concordance between biological duplicates, which were therefore pooled to generate final high-coverage contact matrices for each genotype.
Genome_build: dm6 (Release 6 plus ISO1 MT)
Supplementary_files_format_and_content: cool, validpairs
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| Submission date |
Apr 28, 2021 |
| Last update date |
Feb 01, 2022 |
| Contact name |
Mike Levine |
| E-mail(s) |
msl2@princeton.edu
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| Organization name |
Princeton University
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| Department |
Lewis Sigler Institute
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| Street address |
South Dr
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| City |
Princeton |
| State/province |
NJ |
| ZIP/Postal code |
08540 |
| Country |
USA |
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| Platform ID |
GPL25244 |
| Series (1) |
| GSE173518 |
Transcriptional Coupling of Distant Regulatory Genes in Living Embryos |
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| Relations |
| BioSample |
SAMN18912824 |
| SRA |
SRX10701843 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5269163_kni-knrl-AllGAF1.cool.gz |
161.8 Mb |
(ftp)(http) |
COOL |
| GSM5269163_kni-knrl-AllGAF1_output.pairs.gz |
3.5 Gb |
(ftp)(http) |
PAIRS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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