|
| Status |
Public on Aug 11, 2014 |
| Title |
MNase-seq_CTCF_CP190_RNAi |
| Sample type |
SRA |
| |
|
| Source name |
S2 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: S2
RNAi treatment: DKD (CTCF/CP190 RNAi)
|
| Treatment protocol |
2x10^6 S2 cells were treated with 32µg dsRNA according to the DRSC protocol (http://flyrnai.org/DRSC-PRR.html).
|
| Growth protocol |
S2 cells were grown in Drosophila's Schneider Medium (GIBCO) supplemented with 10% FCS and 1% Penicillin/Streptomycin at 25°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
4x10^7 S2 cells were fixed with 0.3% formaldehyde for 3 min at 18°C with shaking and the reaction terminated by addition of 125 mM glycine. The cell pellet is washed first with PBS and second with 1 ml Nuclei Buffer (10 mM Tris (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, protease inhibitors) and centrifuged after every step at 720 g for 5 min at 4°C. The pellet is resuspended in 1 ml Nuclei Buffer, incubated for 10 min at 4°C and cells disrupted with 20 strokes of a loose pestle. Nuclei were collected by centrifugation as described above and resuspended in 1 ml MNase Buffer (15 mM Tris (pH 7.4), 60 mM KCl, 15 mM NaCl, 1 mM CaCl2, 250 mM sucrose, 0.5 mM DTT,). 200 µl aliquots were treated with 250 U MNase (Thermo Scientific #EN0181) for 40 min at 18°C and afterwards the reaction is stopped with 12.5 mM EDTA and 0.5% SDS. Proteins and RNA were degraded, the resulting DNA purified and electrophoresed on an agarose gel. Mononucleosome bands were excised from gel and processed for sequencing.
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).
|
| |
|
| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
MNase-seq after CTCF/CP190-specific RNAi
|
| 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 MNase accessibility 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
|
| |
|
| 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
|
| Organization name |
Justus-Liebig-University Giessen
|
| Department |
Biomedical Informatics and Systems Medicine
|
| Street address |
Aulweg 132
|
| City |
Giessen |
| State/province |
Hessen |
| ZIP/Postal code |
35392 |
| Country |
Germany |
| |
|
| Platform ID |
GPL11203 |
| Series (2) |
| GSE51599 |
CTCF/CP190 and ISWI dependent regulation of nucleosome occupancy [MNase-seq] |
| GSE51600 |
CTCF/CP190 and ISWI dependent regulation of nucleosome occupancy |
|
| Relations |
| BioSample |
SAMN02381153 |
| SRA |
SRX367044 |
