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| Status |
Public on Oct 30, 2014 |
| Title |
Female_Ovary_DamDsxF_rep1 |
| Sample type |
SRA |
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| Source name |
Adult Drosophila ovary
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: ovary
Sex: Female
age: 5-day old adult
genotype: w1118; UAS-DamDsxF/+
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| Treatment protocol |
We collected replicates for experiments by crossing the male flies bearing UAS-DamDsxF, UAS-DamDsxM or UAS-Dam (serves as control) to female virgin w1118 flies. In order to generate the pUAST-att-NDamMyc-dsxM/F DNA constructs, dsx male (dsxM) and dsx female (dsxF) cDNA sequences were PCR amplified using using sex-specific dsx cDNA plasmid templates (a gift from G. Lee). The forward PCR primer (common to dsx male and female) was Dsx-Dam-F (GTC GAC ATG GTT TCG GAG GAG AAC TGG) and contains a SalI site. The sex-specific reverse primers were DsxM-Dam-R2 (GGT ACC CGG GGA TTA CAC TTT GAT AGT C) and DsxF-Dam-R3 (GGT ACC CAG TTT TGA TAC CCA GAC CC) used to amplify dsxM and dsxF respectively. dsxM and dsxF PCR products were cloned and sequenced in full prior to ligation as SalI/KpnI fragments into the XhoI and KpnI sites of pUAST-att-NDamMyc (a gift from T. Southall) generating plasmids pUAST-att-NDamMyc-dsxM and pUAST-att-NDamMyc-dsxF , respectively. The UAS-Dam, UAS-DamDsxF, or UAS-DamDsxM constructs were inserted independently into the attP2 site on chromosome 3L band 681A-B2 between CG6310 and Mocs1 (Groth et al., 2004 Genetics 166: 1775-1782) using the PhiC31 site-directed integration method. Crosses were set up with 5 virgin females and 3 males. Progeny that were UAS-Dam/+, UAS-DamDsxF/+ or UAS-DamDsxM/+ were collected within 24 hours of eclosion, sexed and aged for 5 days at 25 °C with w1118 flies of the opposite sex. At 5 days of age, flies were dissected in PBS and fatbody or ovaries were collected.
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| Growth protocol |
Flies were raised and maintained at constant light, temperature and humidity (25 ˚C and 60% relative humidity) before DNA extraction. Flies were grown on a cornmeal medium supplemented with Baker's Yeast pellets.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using components of Qiagen's DNEasy Blood and Tissue kit (Qiagen, Valencia, CA, USA). Samples processed for DamID-seq were homogenized in 175 µl PBS and incubated with 200 mg of RNAse A for 2 minutes at room temperature. Tissue was lysed with 20 µl of proteinase K and 200 µl of buffer AL for ten minutes at 70 °C. 200 µl of ethanol were added to each sample and they were transferred to the spin columns after which genomic DNA extraction continued following manufacturer's instructions with the exception of a 30 minute incubation prior to first elution and a second elution step after a 10 minute incubation. Genomic DNA extracted for DamID-chip was extracted following manufacturer's protocol except for the following modifications: a 1.5 hr incubation with lysis buffer prior to the addition of proteinase K, addition of 400 µl of Buffer AL and 300 µl of 100% ethanol, two rounds of both AW1 and AW2 and an incubation with the elution buffer for 30 minutes prior to two rounds of elution. 2.5 - 3 µg of fatbody genomic DNA and 0.3 µg of ovary genomic DNA was used for selective PCR amplification of methylated DNA. DNA was incubated with 10-30 units of DpnI in 50-100 µl of Buffer 4 (New England Biolabs , Ipswich, MA, USA). DpnI was inactivated at 80 °C for 20 minutes and digested DNA was purified through a Qiagen PCR Purification Qiaquick column following manufacturer's protocol and eluted in 30 µl ddH20. One-half of the DpnI reaction products were ligated to 40 pmol of the doublestranded DamID adaptors (top strand : 5'-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3'; bottom strand: 5'-TCCTCGGCCG-3') for 2 hours at 16 °C with 400 units of T4 ligase (New England Biolabs, Ipswich, MA, USA) or 5 units of T4 ligase (Roche, Indianapolis, IN, USA) in a 20 µl reaction volume. All 20 µl of the adapter-ligated DNA were then subjected to DpnII digestion with 10 units of DpnII in a 80 µl reaction volume for at least one hour. PCR amplification was performed with 20 µl of the DpnII digested DNA in an 80 µl volume with 100 pmol PCR primer (5'-TCCTCGGCCG-3'), 16 nmol of each dNTP and 1.6 µl PCR Advantage enzyme mix in 1X PCR Advantage Reaction Buffer (Clontech, Mountain View, CA, USA) or 62.5 pmol PCR primer (5'-TCCTCGGCCG-3'), 16 nmol of each dNTP, 80 nmol MgCl2 in 1X buffer with 8 units of taq polymerase (Fermentas, Pittsburgh, PA, USA). DNA was amplified with the following program: 10 minutes at 68 °C, 1 minute at 94 °C, 5 minutes at 65 °C and 15 minutes at 68 °C, followed by 3 cycles of 1 minute at 94 °C, 1 minute at 65 °C and 10 minutes at 68 °C and then 17 cycles of 1 minute at 94 °C, 1 minute at 65 °C and 2 minutes at 68 °C. DNA was purified through a Qiaquick column (Qiagen, Valencia, CA, USA).
PCR-amplified DNA was sonicated in a 200 µl volume of Qiagen's EB buffer in a BioRuptor Sonicator (Diagenode, Denville, NJ, USA) set on high for 3 X 15 minutes in a 4 °C water bath. Following sonication, DNA was purified through a Qiaquick column (Qiagen, Valencia, CA, USA).
20 ng of sonicated DamID-prepared DNA were used to make libraries following the protocol in the Illumina ChIP-seq Sample Preparation Kit (Illumina, San Diego, CA, USA). A gel slice of 250-350 bp was excised from the gel prior to PCR amplification.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Two biological replicates were collected and sequenced for the treatment condition (UAS-DamDsxF) and control condition (UAS-Dam). Peaks were called by incorporating data from both replicates thus there is one peak file per tissue and sex.
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| Data processing |
Sequencing was done on a HiSeq2000 instrument and base-calls were performed with Illumina software pipeline CASAVA-1.8.0/RTAVersion-1.12.4/HiSeqControl-1.4.5
17 bp were trimmed from either side of the reads before alignment to the genome using Bowtie version 0.12.7 with the parameters -m1 -v2 -5 17 -3 17 --best. Trimming was done to remove primer sequence present in many of the reads and improve % alignment to the genome.
Duplicate reads were removed from the aligned reads using with the Picard tool MarkDuplicates version 1.95.
Following duplicate-removal, reads were counted in 500 bp bins across the genome (GTF file = GSE27269_Dmel_500bp_Tiled.GTF; file available as supplementary data file on the GSE27269 series accession) using HTSeq-count version 0.5.1p2 with parameters -m union -s no
DESeq version 1.12 was used to identify 500 bp bins with statistically significant (adjusted P<0.01) enrichment (log2 Fold Change DamDsx/Dam Only > 0) of DamDsx reads compared to DamOnly reads. Both biological replicates for DamDsx and DamOnly were used to run DESeq with the parameter sharingMode = "fit-only". This step enabled incorporation of both replicates into the process of peak calling. Bins with zero reads in both DamDsx and DamOnly control were removed from the analysis. Peaks were formed by merging adjacent signficantly DamDSX-enriched bins into single units using BEDTools version 2.16.2. We included as peaks all significantly enriched isolated 500 bp bins.
BedGraph files were made using the smoothed tag density function of SPP (version 1.11) with the parameters bandwidth=200, step=100 and tag.shift=0. The score represents the smoothed DamOnly control-subtracted DamDSX tag density.
*_occupancysignal_pvalues.txt files contain the depth-normalized read count averaged between replicates for each 500 bp bin and the associated pvalue and Benjamini-Hochberg adjusted pvalue.BaseMeanA = DamOnly control. BaseMeanB = Dam-Dsx treatment. fold change and log2 fold change represent DamDsx Treatment/DamOnly Control. This file was produced with DESeq v.1.12 as described above.
genome build: dm3, Flybase release 5 with no Uextra
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| Submission date |
Oct 29, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Brian Oliver |
| E-mail(s) |
[email protected]
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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 |
GPL13304 |
| Series (1) |
| GSE27269 |
Identifying targets of DSX with DamID-seq |
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| Relations |
| BioSample |
SAMN02388205 |
| SRA |
SRX370360 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1254262_Ovary_R1.bedgraph.gz |
18.5 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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