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Status |
Public on Oct 30, 2014 |
Title |
S2_DsxM |
Sample type |
SRA |
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Source name |
S2 cells transfected with pMT5.1- DSXM-V5-His B
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Organism |
Drosophila melanogaster |
Characteristics |
chip antibody: anti-V5 (Invitrogen) cell line: S2 treatment: Transfection of S2 cells was performed using Effectene Transfection Reagent (Qiagen, Valencia, CA). pCoBlast (Invitrogen, Carlsbad, CA) was used as the selection plasmid. For the transfection procedure, cells were diluted to a density of 10^6 cells/ml and grew afterwards for about 20 hours. 1 µg expression plasmid, 50 ng pCoBlast, Effectene Transfection components and 840 µl complete Schneider’s Drosophila medium were mixed according to the manufacturer’s protocol and immediately added drop-wise onto the cells. The cells were subsequently grown in complete Schneider’s Drosophila medium for 60 hours prior to selection with 30 µg/ml blasticidin (Invitrogen, Carlsbad, CA). After 5 weeks of selection, the blasticidin-resistant cells were maintained in complete Schneider’s Drosophila medium containing 25 µg/ml of blasticidin. Expression of the recombinant proteins from the MT promoter was induced by adding copper sulfate to the medium to a final concentration of 500 μM. Before induction of expression cells were seeded to a density of 1*106 cells/ ml. Expression of the recombinant proteins was induced at a cell density of 3*10^6 cells/ ml. 60 ml of induced transfected Drosophila S2 cells expressing the recombinant proteins DSXM -V5-His or DSXF -V5-His respectively were grown in cell culture bottles for 36 hours reaching a cell density of 4.5*10^6 cells per ml. amount of dna used to make library: 100 ng
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Growth protocol |
Schneider's Drosophila line 2 cells (abbreviated S2) derived from Drosophila embryos were maintained in Schneider's Drosophila medium (Invitrogen) containing 10% heat inactivated Fetal Bovine Serum (JRH Biosciences) and antibiotics (0.5 U/ml penicillin and 0.5 μg/ml streptomycin (Invitrogen). The cells were maintained in monolayers at 25 °C.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Formaldehyde was added to a final concentration of 1 % to cross-link proteins to DNA. The fixation reaction was incubated on a shaker for 10 min at room temperature. The reaction was quenched by adding glycine to a final concentration of 125 mM and incubation for 5 min on a shaker at room temperature. Subsequently the cells were washed twice with ice-cold PBS. After centrifugation at 500 x g (1680 rpm) for 5 min at 4 °C the cell pellet was resuspended in 10 ml ice-cold cell lysis buffer (5 mM pH8.0 PIPES buffer, 85 mM Potassium chloride, 0.5% Nonidet P40 and protease inhibitors (cOmplete, EDTA-free, Roche, indianapolis, IN)) for 10 minutes at 4 °C. Nuclei were released by douncing with a Wheaton homogenizer (pestle B). The crude nuclear extract was collected by centrifugation at 500 x g (1680 rpm) for 5 min at 4 °C, resuspended in 2 ml ice–cold nuclear lysis buffer (50 mM pH8.1 Tris.HCl, 10 mM EDTA, 1 % SDS and protease inhibitors) and incubated for 20 minutes at 4 °C. After adding 1 ml ice-cold IP dilution buffer (0.01 % SDS, 1.1 % TritonX-100, 1.2 mM pH 8 EDTA.Na2, 16.7 mM pH8 Tris.HCl, 167 mM NaCl and protease inhibitors) and 0.3 g acid-washed glass beads (Sigma-Aldrich, St. Louis, MO) to the nuclear extract, the chromatin was sheared to 200-1000 bp using a Misonix Sonicator 3000 (Misonix, Inc. Farmingdale, NY). Sonication was performed on ice water with 8 pulses of 30 seconds and 30 second intervals. Thereafter the cell debris was removed by centrifugation at 16000 x g (13000 rpm) for 10 min at 4 °C. Aliquots of sonicated chromatin were stored at -80 °C. To assess chromatin quality, aliquots of the chromatin (100 μl) were treated with 40 µg proteinase K (Invitrogen, Carlsbad, CA) for 2 h at 50 °C after the DNA-protein complexes were de-crosslinked at 65°C overnight. After purification of the DNA by phenol-chloroform extraction and ethanol precipitation 3 μl aliquots were subjected to electrophoresis and the chromatin fractions with optimal size (200-1000 bp) range were subjected to immunoprecipitation experiments. Libraries were made using the Genomic DNA sample preparation kit (Illumina). Adapter-ligated DNA of 200 ± 25 bp range was excised from the gel before PCR amplification.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina Genome Analyzer |
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Description |
Three biological replicates were produced. Each fastq file for S2_DSM represents an independent biological replicate. All fastq files for S2_DsxM were pooled before alignment to the genome.
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Data processing |
Sequencing was done on a GAI instrument and base-calls were performed with Illumina software pipeline version 0.3.0
All reads were trimmed to 25 bp before alignment to the genome using Bowtie version 0.12.7
Peaks were called using SPP version 1.11 WTD method with FDR of 0.01. Duplicate reads were removed from the libraries before peak calling with the Picard tool MarkDuplicates (version 1.95)
Bedgraph files for S2_DSXF and S2_DsxM (input subtracted) were made using SPP's smoothed tag density function with bandwidth=200 and step=100 and tag.shift=0. The score represents the smoothed input subtracted tag density.
Genome_build: dm3, Flybase release 5 with no Uextra
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Submission date |
Aug 02, 2013 |
Last update date |
May 15, 2019 |
Contact name |
Brian Oliver |
E-mail(s) |
briano@nih.gov
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Phone |
301-204-9463
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Organization name |
NIDDK, NIH
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Department |
LBG
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Lab |
Developmental Genomics
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Street address |
50 South Drive
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City |
Bethesda |
State/province |
MD |
ZIP/Postal code |
20892 |
Country |
USA |
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Platform ID |
GPL9058 |
Series (2) |
GSE49480 |
Identifying targets of DSX with ChIP-seq, DamID-seq and DamID-chip and transcriptional response to DSX isoform switch |
GSE49511 |
Identifying Targets of DSX with ChIP-seq |
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Relations |
BioSample |
SAMN02299538 |
SRA |
SRX330996 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1200248_S2_DsxM.bedgraph.gz |
17.8 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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