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Sample GSM1545891

Query DataSets for GSM1545891

Status

Public on Nov 18, 2014

Title

Dmel_Dam_rep1

Sample type

SRA

Source name

whole embryos, 0-16 hours

Organism

Drosophila melanogaster

Characteristics

strain of origin: w[1118]
transgene inserted: pBac3xP3-EGFP-Dam
original genome build: dm3 (UCSC)
translated genome build: NA

Treatment protocol

Each line of flies was transformed with a piggyBac plasmid containing either a Dam-only transgene or a Dichaete-Dam or SoxNeuro-Dam fusion transgene. Transgenic flies were identified by the presence of GFP expression in the eyes.

Growth protocol

Flies were grown on standard cornmeal medium at 25 degrees C, with the exception of D. pseudoobscura, which were grown on banana-opuntia-malt medium at 22.5 degrees C. Embryos were collected after overnight lays on a grape juice-agar plate streaked with fresh yeast paste.

Extracted molecule

genomic DNA

Extraction protocol

To extract high-molecular weight genomic DNA, each aliquot of embryos was dechorionated and homogenized in a Dounce 15-ml homogenizer in 10 ml of homogenization buffer (10 mM Tris-HCl pH 7.6, 60 mM NaCl, 10 mM EDTA, 0.15 mM spermine, 0.15 mM spermidine, 0.5% Triton X-100). The lysate was then spun for 10 minutes at 6000g. The supernatant was discarded, and the pellet was resuspended in 10 ml homogenization buffer, then spun again for 10 minutes at 6000g. The supernatant was again discarded, and the pellet was resuspended in 3 ml homogenization buffer. 300 μl of 20% n-lauroyl sarcosine were added, and the samples were inverted several times to lyse the nuclei. The samples were treated with RNaseA followed by proteinase K, then purified by two phenol-chloroform extractions and one chloroform extraction. DamID samples were prepared from genomic DNA following the DamID protocol from Vogel et al. (Nat Protoc 2, 1467-1478, 2007).
Libraries were prepared as for ChIP-seq by BGI Tech, starting with at least 20 ng of DNA per sample and following standard Illumina protocol (end repair, 3' dA-tailing, ligation of adapters, PCR amplification and size selection for fragments 100-300bp). Libraries were multiplexed with 2 samples per run for the MiSeq and 9-12 samples per lane for the HiSeq. MiSeq libraries were run as 150-bp paired-end reads, while HiSeq libraries were run as 50-bp single-end reads.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina MiSeq

Description

DamID-derived DNA
Replicate 1 of Dam-only (control) in D. melanogaster

Data processing

DamID adapters were removed from 5' and 3' ends of reads using cutadapt with the following command: cutadapt -a GATCCTCGGCCGCGACC -g ^GGTCGCGGCCGAGGATC -o sample_cutadapt.fq.gz sample.fq.gz
Reads were mapped to their respective original genomes using bowtie2 with the default parameters.
Mapped reads were converted to bam format, sorted and indexed using SAMtools version 0.1.19.
BEDTools version 2.21.1 was used to convert reads to bed format (bamToBed), and the BEDTools slop utility was used to extend reads to the average library fragment size.
For samples from all non-melanogaster species, UCSC LiftOver was used to translate reads into the dm3 genome assembly. The -minMatch parameter was set to 0.7 for D. simulans and D. yakuba, and it was set to 0.5 for D. pseudoobscura. In all cases, multiple outputs were not permitted.
The position of all GATC fragments in each genome was found using the scanMotifGenomeWide.pl utility from HOMER v4.7. The BEDTools coverage utility was used to count the number of reads overlapping each GATC fragment in each sample, and resulting counts were combined into a count table including all sample and control replicates for each comparison of a Dam-fusion protein vs. Dam-only.
DESeq2 v.1.6.1 was used to test for differential enrichment of Dam-fusion reads versus Dam-only reads in each GATC fragment. Fragments flagged as differentially enriched (log2 fold change >0 and adjusted p-value <0.05 or <0.01) were extracted, and neighboring GATC fragments with less than 100 bp separating them were merged to form binding intervals using an in-house perl script.
Binding profiles were created by using an in-house perl script to extract the normalized log2 fold changes between the fusion protein and Dam-only control calculated by DESeq2 for each GATC fragment.
Genome_build: Varies per sample (dm3, droSim1, droYak2, dp3)
Supplementary_files_format_and_content: Bed files contain the locations of binding intervals (described above). Column 4 shows the number of neighboring GATC fragments that were merged to create each interval. For D. melanogaster, binding intervals called at FDR1 and FDR5 are provided. For all non-melanogaster species, binding profiles are provided for data in the original genome (untranslated) and data that has been translated to the D. melanogaster dm3 genome assembly.
Supplementary_files_format_and_content: Bedgraph files contain binding affinity profiles, which are the normalized log2 fold changes between the fusion protein and Dam-only control for each GATC fragment across the genome. For all non-melanogaster species, binding profiles are provided for data in the original genome (untranslated) and data that has been translated to the D. melanogaster dm3 genome assembly.

Submission date

Nov 17, 2014

Last update date

May 15, 2019

Contact name

Sarah Carl

E-mail(s)

sarah.carl@fmi.ch

Organization name

Friedrich Miescher Institute

Department

Epigenetics