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| Status |
Public on Mar 17, 2016 |
| Title |
BR.repo.min.Dam_DamIDSeq_rep1 |
| Sample type |
SRA |
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| Source name |
repo+ glial cells of larval central brain
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| Organism |
Drosophila melanogaster |
| Characteristics |
gender: pooled male and female
genotype: y,w/w; repoFlp5/M{min.hsp70P-STOP#1-Dam}ZH51C
developmental stage: wandering third instar larvae
tissue: brain without ventral nerve cord
dam-fusion protein: Dam only
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| Treatment protocol |
Central brains were dissected from 20 larvae in 1×PBS [140 mM NaCl, 10 mM PO4 Buffer, 3 mM KCl; Invitrogen cat. no. 18912-014] using forceps and dissection needles and collected in 100 μl TENS solution [100 mM Tris-HCl (pH 8.0); 5 mM EDTA (pH 8.0); 200 mM NaCl; 0.2% SDS] at room temperature.
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| Growth protocol |
Flies were raised at 25°C on standard cornmeal/molasses/agar medium.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Two μl of Proteinase K (20 mg/ml) [Roche, 03115887001] were added to dissected central brains and the sample was incubated at 65°C for 6 hours. Next, 1 μl of RNase A (100 mg/ml) [QIAGEN, 1007885] was added and the sample was incubate at 37°C for 30 minutes. The lysate was transferred to a pre-spun (1 minute at 14,000 rpm just prior to use) phase lock gel light 1.5ml tube [5prime, 2302800] and 100 μl of phenol:chloroform:isoamylalcohol (25:24:1) [Sigma-Aldrich, P2069] were added. The sample was mixed well by shaking and the tube was spun for 5 minutes at 14,000 rpm at room temperature. The upper phase (~90 μl) was transferred into a new tube. To precipitate DNA, 1 µl of glycogen (20 mg/ml) [Fermentas, R0561], 10 µl of 3M NaAc (pH 5.2) and 300 µl of ethanol were added, mixed well by inversion and the solution was kept overnight at -20°C. The tube was spun for 30 minutes at 14,000 rpm at 4°C. The supernatant was removed and the pellet was washed with 500 µl of ice cold 70% ethanol. The tube was spun for 5 minutes at 14,000 rpm at 4°C. The supernatant was carefully removed and the pellet was air dried for 15 minutes and dissolved in 12 µl of double distilled water. DNA concentration was measured using a NanoDrop spectrophotometer. Dam-methylated fragments were amplified as described previously (Vogel, M.J. et al. Detection of in vivo protein-DNA interactions using DamID in mammalian cells. Nat Protoc 2(6), 1467-78, 2007) using 500 ng of DNA as an input.
The amplified methylated DNA fragments were purified using QIAquick PCR Purification kit (QIAGEN, 28104) according to the manufacturer’s instructions. DNA was eluted in nuclease-free water (Ambion, AM9938). From 200 ng to 3 µg of the purified PCR products were diluted in 100 μl of nuclease-free water and transferred into a snap-cap microTUBE (6 × 16 mm) with AFA fiber (Covaris, 520045). DNA was sheared using the Covaris S2 instrument at the following settings: duty cycle = 10%, intensity = 5, cycles/burst = 200, time = 45 sec (as consequence, power = ~23W and bath temperature = ~4°C). The size of the DNA fragments before shearing was usually a smear in the range of 250–2000 bp with a peak at 600 bp, after shearing this was reduced to become a range of 100–500 bp with a peak around 300 bp. After shearing, the DNA was purified and concentrated using Agencourt magnetic beads (Beckman Coulter, A63881; 160 µl of beads were added to 100 µl of sheared DNA solution). DNA was eluted in 50 µl of nuclease-free water. DNA concentration was measured using an Agilent BioAnalyzer 2100 DNA 7500 chip (Agilent, 5067-1506). One μg of sheared material was used to prepare Illumina sequencing library with TruSeq DNA HT Sample Prep Kit (Illumina, FC-121-2003) according to the manufacturer’s instructions. Seven cycles of PCR were performed. This step included sample indexing. The library was quantified on an Agilent BioAnalyzer 2100 DNA 7500 chip that lists the product size distribution and quantifies the concentration in µg/µl and in nM.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
DamID system used: DamFLP
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| Data processing |
Library strategy: DamID-Seq
Basecalling and filtering were performed using standard software of the Illumina HiSeq 2000.
The 51bp reads were first parsed (using custom scripts and cutadapt (Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17.1,10-2, 2011; http://dx.doi.org/10.14806/ej.17.1.200)) to extract the genomic DNA. The genomic DNA sequences were aligned to the reference genome (dm3) using bowtie2 with default parameters. Reads that did not align or aligned more than once were discarded. Also reads with a MAPQ score of less than 25 were discarded. Reads were then summed per GATC fragment, using HTSeq (Anders, S. et al. HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31(2), 166-9, 2015). Next, the chromosomes “U”, “M” and “Uextra” were excluded from the subsequent analysis.
For the biological samples which were generated using multiple technical replicates ( uninduced Dam only controls from larval central brains), the read counts were summed over the technical replicates. Next, the counts were normalized to the total number of reads per sample. For each biological replicate of the same tissue or cell type (larval central brain, larval fat bodies and repo+ glial cells of larval central brain), the normalized DamID scores were calculated as the log2 of the ratio of the Polycomb and Dam only control. Finally, the normalized DamID scores were averaged over the 2 biological replicates.
Genome_build: dm3
Supplementary_files_format_and_content: tabular text files, containing columns for GATC-fragment-ID, chromosome, start, end, DamID scores
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| Submission date |
Dec 09, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Bas van Steensel |
| E-mail(s) |
[email protected]
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| Phone |
+ 31 20 512 2040
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| Fax |
+31 20 669 1383
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| URL |
http://www.nki.nl/nkidep/vansteensel
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| Organization name |
Netherlands Cancer Institute
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| Department |
division of Molecular Biology
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| Lab |
van Steensel group
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| Street address |
Plesmanlaan 121
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| City |
Amsterdam |
| ZIP/Postal code |
1066 CX |
| Country |
Netherlands |
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| Platform ID |
GPL13304 |
| Series (2) |
| GSE75831 |
Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila [DamID-seq] |
| GSE75835 |
Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila |
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| Relations |
| BioSample |
SAMN04327340 |
| SRA |
SRX1472404 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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