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| Status |
Public on Jan 01, 2018 |
| Title |
input |
| Sample type |
SRA |
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| Source name |
unstaged embryos
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| Organism |
Drosophila melanogaster |
| Characteristics |
developmental stage: embryos
genotype: wt
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Unstaged embryos (1 g) were dechorinated in 120 ml, 1:5 diluted, sodium hypochloride (VWR, Cat.no. 301696S) for 3 min. The embryos were thoroughly washed and fixed in the fixing solution [10 ml, 0.1 M NaCl, 0.05 M HEPES pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 3.7% Formaldehyde (Sigma, Cat. No F1635) added to 30 ml n-Heptane (VWR, Cat. No. 8.22332.1000)] for 15 min at 16-18°C on a rotating wheel. Fixation was quenched by adding 125 mM glycine. The embryos were subsequently washed with PBS (including 0.01% Triton-X100) for 10 min and stored at -80°C until further use. For nuclei isolation, embryos were slowly thawed and dounced using a glass homogenizer (Schubert, Cat.no. 9164693) with 20 strokes each of the A and B pestles in ice-cold NX-I buffer [15 mM HEPES pH 7.6, 10 mM KCl, 2 mM MgCl2, 0.5 mM EGTA, 0.1 mM EDTA, 350 mM sucrose, 1 mM DTT, 0.2 mM PMSF, Protease inhibitors Leupeptin, Pepstatin and Aprotinin (10 µg/ml)]. The lysate was filtered through Miracloth and nuclei were pelleted at 3500 rpm, 10 min, 4°C. The pellet was washed in NX-I buffer. Finally, the nuclei were resuspended in RIPA [1% Triton X-100, 0.1% Sodium deoxycholate, 140 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.1% SDS, 1 mM PMSF] and washed 3 times. Nuclei were then counted and frozen at -80°C in aliquots of ~109 nuclei/ml. For shearing and ChIP, thawed nuclei were adjusted to ~2x108/ml by dilution in RIPA and sheared with a Covaris S220 system (Covaris Inc. MA, USA) at 110 Watts, 20% duty factor and 200 cycles per burst for 25 min. Chromatin was pre-cleared using protein a A+G bead (1:1) mix for 1h at 4°C. Immunoprecipitations were set up overnight at 4°C with 200 µl chromatin and 4 µl of the respective antibody adjusted to 500 µl with RIPA. RIPA-equilibrated protein A+G (1:1) mix was then added to precipitate the immune-complexes for 3 h at 4°C. Beads were washed subsequently 5 times for 10 min each in 1 ml RIPA buffer. Residual RNA was digested by RNase A (10 µg/100 µl, Sigma, Cat. No. R4875) at 37°C for 20 min. Subsequent protein digestion (25 µg/100 µl, Proteinase K, Genaxxon, Cat.no. M3036.0100) and reversal of cross-linking were performed together at 68°C for 2 hours. DNA was purified using GenElute™ PCR Clean-Up Kit (Sigma, Cat.no NA1020)
ChIP DNA was quantified using the Qubit® dsDNA HS Assay Kit (Life Technologies, Cat.no.Q32851) and sequencing libraries were prepared using the MicroPlex Library Preparation kit (Diagenode, Cat. No. C05010011) starting from 2 ng DNA, whenever possible. PCR amplification was monitored by quantifying amplified libraries (maximum 19 cycles).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Alignment to dm3 without chromosome Uextra using bowtie version 1.1.1 (parameter -m 1)
Read extension to 200 bp and coverage vector calculation, bedGraph files were generated from coverage vectors using export.bedGraph (bioconductor package 'rtracklayer')
Peak calling with Homer. CTCF called over input (parameters: -style factor -size 200 -fragLength 200 -inputFragLength 200). H2A.V callled over H3 ChIP (parameters: -style histone)
Genome_build: dm3
Supplementary_files_format_and_content: bedgraph files of coverage and bed files of ChIP-peaks
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| Submission date |
Aug 09, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Tobias Straub |
| E-mail(s) |
tstraub@med.uni-muenchen.de
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| Organization name |
LMU Munich
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| Department |
Biomedical Center, Bioinformatics
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| Street address |
Großhadener Str. 9
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| City |
Martinsried |
| ZIP/Postal code |
82152 |
| Country |
Germany |
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| Platform ID |
GPL19951 |
| Series (2) |
| GSE85404 |
ACF1 regulates nucleosome spacing in Drosophila euchromatin [ChIP-seq] |
| GSE85407 |
ACF1 regulates nucleosome spacing in Drosophila euchromatin |
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| Relations |
| BioSample |
SAMN05554098 |
| SRA |
SRX2010896 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2266305_B40_Input_LJan14_DJ_cov.bedGraph.gz |
193.4 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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