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Sample GSM1333888 Query DataSets for GSM1333888
Status Public on Dec 02, 2014
Title Dorsal ChIP-nexus (Drosophila embryos) Rep 2
Sample type SRA
Source name Drosophila melanogaster_whole-embryo
Organism Drosophila melanogaster
Characteristics tissue: whole-embryo
time point: 02 to 04 h AEL
chip antibody: anti-Dorsal rabbit polyclonal, custom (GenScript)
strain: Oregon-R
Treatment protocol no treatment, only fixation by formaldehyde
Growth protocol D. melanogaster embryos were collected and aged on yeasted apple juice plates at 25C and 60 % humidity
Extracted molecule genomic DNA
Extraction protocol embryos were cross-linked in 1.8% formaldehyde. Embryos were then dounced to break down cells and extract nuclei. Chromatin was sonicated by bioruptor to an average size of 300~500 bp. 300 ul soluble chromatin from ~100 mg embryos was used
DNA-protein-complexes were isolated with specific antibodies when still being attached to protein A-dynabeads. DNA was end repaired using T4 DNA polymerase, DNA polymerase I large fragment (Klenow polymerase) and T4 polynucleotide kinase. A single 3'-A overhang was added to the blunted ends using Klenow 3'-5' exo- polymerase. Adapters with single 3'-T overhangs and 5’ overhangs (barcode composed of 5 random nucleotides) were ligated to the adenylated fragments. Ligated fragments were blunted again by Klenow 3'-5' exo- polymerase to fill in the 5’ overhang first and then by T4 DNA polymerase to trim any possible 3’ overhang. Blunted DNA was subsequently digested by lambda exonuclease first and RecJf afterwards. Digested single strand DNA was then eluted, reverse cross-linked and purified prior to self-circularization by Circligase. An oligonucleotide was mixed with circularized single DNA for subsequent BamHI digestion to linearize the single DNA again. Linearized single strand DNA was then PCR-amplified using adaptor sequences and library was purified on 2% agarose gel to remove adaptor-adaptor ligation products. Following cluster generation on the flowcell surface, the sequencing libraries were sequenced following Illumina's protocol.
Library strategy ChIP-Seq
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2000
Description dmel_embryo_dl_chipnexus_02
Illumina multiplex barcode: TGACCA
Data processing Base calling, read filtering and demultiplexing were performed by Illumina CASAVA 1.8.2 with default settings
Custom barcode sequences were removed from the first 9bp of each read and sequencing adapters were trimmed from the 3' end using cutadapt v1.3
Remaining reads with length >= 22bp were aligned to the reference genome using bowtie v1.0.0, retaining only uniquely aligning reads with a maximum of 2 mismatches
Aligned reads were separated by strand and reduced to a single base at the 5' end
Genome-wide coverage counts were calculated for each strand separately and saved in BigWig format
Supplementary_files_format_and_content: BigWig files contain genome coordinates and counts of the first base of aligned reads, separated by alignment strand
Genome Build: UCSC dm3 UCSC dm3
Submission date Feb 24, 2014
Last update date May 15, 2019
Contact name Julia Zeitlinger
Organization name Stowers Institute for Medical Research
Lab Zeitlinger Lab
Street address 1000 E 50th St
City Kansas City
State/province MO
ZIP/Postal code 64110
Country USA
Platform ID GPL13304
Series (1)
GSE55306 ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints
BioSample SAMN02650938
SRA SRX475441
Named Annotation
Named Annotation

Supplementary file Size Download File type/resource 22.4 Mb (ftp)(http) BW 22.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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