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| Status |
Public on Aug 11, 2014 |
| Title |
H3_ChIPseq_CTCF_CP190_RNAi |
| Sample type |
SRA |
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| Source name |
S2 cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: S2
RNAi treatment: DKD (CTCF/CP190 RNAi)
chip antibody: H3
chip antibody vendor: Abcam
chip antibody cat. #: ab1791
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| Treatment protocol |
2x10^6 S2 cells were treated with 32µg dsRNA according to the DRSC protocol (http://flyrnai.org/DRSC-PRR.html).
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| Growth protocol |
S2 cells were grown in Drosophila's Schneider Medium (GIBCO) supplemented with 10% FCS and 1% Penicillin/Streptomycin at 25°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
S2 cells were fixed in 1% formaldehyde for 15 min at 18°C. The reaction was stopped by adding glycine to a final concentration of 125 mM. After washing, cells were resuspended in SDS Lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1), protease inhibitors) and incubated 10 min on ice. Chromatin was prepared by shearing with Bioruptor (Diagenode) at setting “high” for 20 cycles with 30 sec “On” and 30 sec “Off” to yield chromatin with a size ranging from 200-800 bp. The sonicated chromatin was diluted 10 fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl, protease inhibitors). Chromatin was immunoprecipitated by incubating with the appropriate antibodies overnight at 4°C. To collect the antibody/chromatin complexes the solution is incubated for 1 h at 4°C with 30 µl Protein G Plus/Protein A Agarose Suspension (Millipore). After washing one time with Low Salt Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl), one time with High Salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl), one time with LiCl Buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)) and two times with TE Buffer (10 mM Tris-HCl, 1 mM EDTA (pH 8.0)) the crosslinks were reversed and DNA is recovered by illustra GFX columns (GE Healthcare)
Sequencing libraries were prepared from 10 ng of immunoprecipitated DNA with the Illumina ChIPSeq DNA Sample Prep Kit according to Illuminas instructions. Cluster generation was performed using the Illumina cluster station, sequencing on the Genome Analyzer IIx followed a standard protocol. The fluorescent images were processed to sequences using the Genome Analyzer Pipeline Analysis software 1.8 (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
H3 ChIP-seq after CTCF/CP190-specific RNAi
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| Data processing |
Quality control of FASTQ files was done using FastQC.
Removal of low quality reads was done using fastx_toolkit-0.06
Reads were aligned to a pre-compiled index of the mouse hg19 genome using Bowtie (options: -k 1 -m 1) 0.12.5.
Duplicate reads were removed with Samtools' rmdup function.
Samtools was used to convert aligned reads to BAM format.
For Browser visualization wiggle tracks were generated using MACS 1.4 at 10 bp resolution
In order to determine the effect of RNAi treatments on H3 density the dm3 genome was binned into 100 bp bins. For each condition the total number of overlapping reads was determined. Next we used DESeq in order to correct for differential library sizes and to calculate fold enrichment/depletion per bin between two conditions (e.g. ISWI RNAi versus luciferase RNAi).
Genome_build: dm3
Supplementary_files_format_and_content: Wiggle (WIG) files were produced using Macs 1.4 at 10 bp resolution
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| Submission date |
Oct 23, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Marek Bartkuhn |
| E-mail(s) |
marek.bartkuhn@gen.bio.uni-giessen.de
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| Organization name |
Justus-Liebig-University Giessen
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| Department |
Biomedical Informatics and Systems Medicine
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| Street address |
Aulweg 132
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| City |
Giessen |
| State/province |
Hessen |
| ZIP/Postal code |
35392 |
| Country |
Germany |
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| Platform ID |
GPL11203 |
| Series (2) |
| GSE51598 |
CTCF/CP190 and ISWI dependent regulation of nucleosome occupancy [ChIP-seq] |
| GSE51600 |
CTCF/CP190 and ISWI dependent regulation of nucleosome occupancy |
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| Relations |
| BioSample |
SAMN02381146 |
| SRA |
SRX367040 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1248972_H3_dkd_dm3.wig.gz |
25.4 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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