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| Status |
Public on Oct 30, 2014 |
| Title |
FemaleFatbody_DamOnly_R1 |
| Sample type |
genomic |
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| Source name |
Female_Fatbody_DamOnly
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| Organism |
Drosophila melanogaster |
| Characteristics |
Sex: Female
tissue: Fatbody
age: 5 day-old adult
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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).
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| Label |
cy5
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| Label protocol |
Labeling was performed by NimbleGen.
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| Hybridization protocol |
Hybridization was performed by NimbleGen. DamDsxF and DamOnly samples for each replicate were hybridized to the same array. For example, the samples FemaleFatbody_DamDsxF_R1 and FemaleFatbody_DamOnly_R1 were hybridized to the same array.
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| Scan protocol |
Scanning was performed by NimbleGen.
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| Description |
Three biological replicates were collected for treatment (UAS-DamDsxF) and control (UAS-Dam). One Dye Flip was performed.
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| Data processing |
The Series supplementary file "FemaleFatbody_DsxF_DamIDarray_Normalized_Data.txt" contains the normalized probe intensities, DamDsxF and DamOnly means, log2 Fold Change (DamDsxF/DamOnly), pvalues from a ttest and Benjamini-Hochberg adjusted p-values for the ttest. This file also contains the genomic coordinates used for the probes. For this analysis, we forced all probe lengths to be identical. To do this, we used the actual probe start position and added 50 bp (the median probe length on the array) to the start position to make the end position. The actual probe coordinates can be obtained from the .GFF files available on the series entry. Probe intensities were quantile normalized using the R package PreProcessCore (Ben Bolstad) available through BioConductor. A cut-off for the signal detection limit was determined by calculating the mean of all the random probes on the array. The 95th percentile of the mean random probe signal was used as the signal cutoff. For these arrays, the 95th percentile of the mean random probes was 364. Any probe where both DamDsxF and DamOnly mean intensities were 364 or below were considered below detection level and removed from the dataset. All calculations were performed in R. The data present in"FemaleFatbody_DsxF_DamIDarray_Normalized_Data.txt" are the data that were used to define DSXF occupied regions (peaks). Peaks were identified by selecting probes that had an adjusted p-value at or below 0.01 and a positive log2 Fold Change. Significant probes were considered members of a single peak when they occurred within 1 kb of each other. BEDTools v.2.16.2 was used to combine significant probes present within a 1 kb window into peaks.
The Scaled Log2Ratio Data files (.GFF) were provided by NimbleGen. We did not use these files for analyzing the data in our study but are providing them so they will be available to others. This file contains the ratio of the input signals for the experimental and test samples that were co-hybridized to the array. The log2 ratio is computed and scaled to center the ratio data around zero. Scaling is performed by subtracting the biweight mean for the log2-ratio values for all features on the array from each log2-ratio value. View log2-ratio data files (.GFF) using SignalMap software. They can also be opened in a text editor.
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| Submission date |
Aug 01, 2013 |
| Last update date |
Oct 30, 2014 |
| 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 |
GPL10639 |
| Series (2) |
| GSE49478 |
Identifying targets of DSX with DamID-chip |
| GSE49480 |
Identifying targets of DSX with ChIP-seq, DamID-seq and DamID-chip and transcriptional response to DSX isoform switch |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1199839_FemaleFatbody_DamOnly_R1_61488205_635.pair.gz |
32.3 Mb |
(ftp)(http) |
PAIR |
| Processed data are available on Series record |
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