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| Status |
Public on Jan 20, 2012 |
| Title |
[E-MTAB-652] PH-ChIP d5 |
| Sample type |
SRA |
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| Source name |
PH-ChIP d5
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| Organism |
Drosophila melanogaster |
| Characteristics |
material type: cell
cellline: Schneider S2
Sex: male
immunoprecipitate: CID island
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| Treatment protocol |
specified_biomaterial_action | Cells were transfected with FuGENE 6 Transfection Reagent (Roche) and stable lines were selected with 100 mg/ml Hygromycin-B as previously described 25 or in case of co-transfection with pCoPuro with 100mg/ml puromycin. Cells containing stably integrated arrays of Lac Operators on chromosome 3R were obtained by co-transfection with pAFS51 56 and pCoPuro, followed by subsequent cloning of the cell line. Pulse-overexpression of CID and pulse-co-overexpression of CID and HP1 were induced using the metallothionein promoter (pMT/V5 vectors) with 500 mM CuSO4 for 48 h (start day -2). At day 0 Fluorescent Activated Cell Sorting (FACS) was used to select the cells with the highest levels of GFP expression (>75% of max.) on a MoFlo sorter using 70 mm 11 diameter nozzle at 60 psi. Cells were immediately plated in fresh medium lacking inducing reagents for the chase. For HDAC inhibition cells were incubated with 75nM Trichostatin A (Cell Signaling Technology, Inc) from the beginning of CIDGFP pulse (day -2).
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| Growth protocol |
grow | S2 cells were grown in Schneiders Drosophila medium (Serva) supplemented with 10% fetal calf serum and antibiotics (0.3 mg/ml penicillin, 0.3 mg/ml streptomycin and 0.75 ug/ml amphotericin B).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
nucleic_acid_extraction | 4x107 S2 cells in 40 ml Schneider's medium were fixed for 10 minutes at room temperature by addition of 1% formaldehyde. After 2 ice-cold washes in PBS, cells were lysed in 2.6ml of L2 buffer (L2: 1% SDS, 5mM EDTA, 50mM Tris pH8). The lysate was sonicated to fragment genomic DNA to an average length of around 500 bp, and then diluted 10-fold in DB (DB: 0.5 % NP40, 5 mM EDTA, 200 mM NaCl, 50 mM Tris pH8). Anti-mouse IgG M280 magnetic beads were coated with mouse monoclonal anti-GFP antibody (clone 496) by rotating together for 6 hours at 4C (80mg antibody for 160 ml M280 slurry), followed by washing in DB. For each immunoprecipitation, 160 ml slurry of anti-GFP-coated beads were added to the sonicated lysate from 107 cells and rotated overnight at 4C. Beads were washed once quickly and then 3x for 5 minutes on ice in WB (WB: 0.1% SDS, 1% NP40, 2mM EDTA, 500mM NaCl, 20mM Tris pH8), followed by 3x for 5 minutes on ice in TE (TE: 1mM EDTA, 10mM Tris pH8). Precipitated chromatin was eluted from the beads, and fixation reversed, by incubation overnight at 65C in 60ml EB (EB: 2% SDS, 1mM EDTA, 10mM Tris pH8). DNA was purified using a Qiagen MinElute clean-up kit, and eluted in 30ml (elution buffer: 10mM Tris pH8.5).
sequencing | paired-end strategy (2x36bp)
data_generation | Base calls were done by Solexa/Illumina software pipeline. SRF files were created from the Illumina qseq files with illumina2srf from the sequenceread package version 2.1.4 (options: -P -N '<run id>:%l:%t:' -n '%x:%y').
alignment | The bowtie software was used to align reads against the Dm5.30 reference genome. Up to two mismatches in each read were allowed. The options for bowtie were: -S -p 20 -v 2 -X 600 --tryhard -m 1 --best --strata
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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 |
Performer: MAX-PLANCK-INSTITUTE
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| Data processing |
processed data not provided
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| Submission date |
Jul 27, 2011 |
| Last update date |
May 15, 2019 |
| Organization |
European Bioinformatics Institute |
| E-mail(s) |
miamexpress@ebi.ac.uk
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| Lab |
ArrayExpress
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| Street address |
Wellcome Trust Genome Campus
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| City |
Hinxton |
| State/province |
Cambridgeshire |
| ZIP/Postal code |
CB10 1SD |
| Country |
United Kingdom |
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| Platform ID |
GPL11203 |
| Series (1) |
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| Relations |
| SRA |
ERX012711 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data not provided for this record |
| Raw data are available in SRA |
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