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Sample GSM6757746 Query DataSets for GSM6757746
Status Public on Dec 20, 2022
Title OreR, Cad, nexus, 1
Sample type SRA
 
Source name embryo
Organism Drosophila melanogaster
Characteristics tissue: embryo
genotype: Wild-type (Oregon-R)
developmental stage: 2-3 hours after egg laying
chip antibody: Custom GenScript Cad (aa 1-214)
Treatment protocol All embryos were dechorionated with 50% bleach for 2 minutes, rinsed with water, and fixed with 1.8% formaldehyde while vortexing for 15 minutes. The vitelline membrane was removed using methanol/heptane and embryos were stored in methanol at -20°C until use.
Growth protocol All embryos were collected from population cages using apple juice plates with yeast paste, following two pre-clearings. For ChIP experiments, embryos were collected for 1 hour and aged for 2 hours at 25°C.
Extracted molecule genomic DNA
Extraction protocol For ChIP, 0.2-0.4 grams of fixed 2-3 h AEL embryos were used for all experiments. Chromatin extracts were prepared by douncing embryos in Lysis Buffer A1 (15 mM HEPES pH 7.5, 15 mM NaCl, 60 mM KCl, 4 mM MgCl2, 0.5% Triton X-100, 0.5 mM DTT (add fresh)), washing nuclei with ChIP Buffer A2 (15 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.5% N-lauroylsarcosine, 0.1% sodium deoxycholate, and 0.1% SDS), and sonicating with a Bioruptor Pico (Diagenode) for six cycles of 30 seconds on and 30 seconds off. All ChIP-nexus experiments were performed using antibodies custom generated by Genscript: Zelda (aa 1117-1327), Dorsal (aa 39-346), Twist (C-terminus), Bicoid (C-terminus), Caudal (aa 1-214), GAF (aa 1-382). ChIP-seq experiments were performed with the following commercially available antibodies: H3K27ac (Active motif, 39133) and H3K4me1 (Active motif, 39635).
ChIP-nexus was performed according to He et al., 2015, except that the ChIP-nexus adapter mix contained four fixed barcodes and PCR library amplification was performed directly after circularization of the purified DNA fragments (without addition of the oligo and BamHI digestion). ChIP-seq was performed as previously described in He et al., 2011, and included a whole cell extract (WCE).
ChIP-nexus for transcription factors and ChIP-seq for histone modifications using standard Illumina protocols.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
 
Data processing ChIP-nexus reads were pre-processed by trimming off fixed and random barcodes and reassigning them to FASTQ read names.
ChIP-nexus adapter fragments were trimmed from the 3’ end of the fragments using cutadapt (v.2.5)
ChIP-nexus reads were aligned using bowtie2 (v.2.3.5.1) to the Drosophila melanogaster genome assembly dm6. Aligned ChIP-nexus BAM files were deduplicated based on unique fragment coordinates and barcode assignments.
Genome-wide coverage was calculated and saved in BigWig format.
ChIP-nexus peaks were called using MACS2 (v.2.2.7.1) with parameters designed to resimulate the full fragment length coverage rather than the single stop base coverage (--keep-dup=all -f=BAM --shift=-75 --extsize=150).
ChIP-seq reads were aligned using bowtie2 (v.2.3.5.1) to the Drosophila melanogaster genome assembly dm6. Aligned ChIP-seq BAM files were deduplicated based on unique fragment coordinates and fragments extended based on the average experiment fragment length as determined with an Agilent 2100 Bioanalyzer.
Genome-wide coverage was calculated and saved in BigWig format.
ChIP-seq peaks were called using MACS2 (v.2.2.7.1) with default parameters and an applied background coverage using the associated WCE ChIP-seq control experiment.
Assembly: UCSC dm6
Supplementary files format and content: BigWig, narrowPeak (except for Input sample)
 
Submission date Nov 27, 2022
Last update date Dec 23, 2022
Contact name Kaelan Joseph Brennan
E-mail(s) KBrennan@stowers.org
Phone 8125281836
Organization name Stowers Institute for Medical Research
Lab Zeitlinger
Street address 1000 East 50th Street
City Kansas City
State/province MO
ZIP/Postal code 64110
Country USA
 
Platform ID GPL19132
Series (2)
GSE218849 Chromatin accessibility in the Drosophila embryo is determined by transcription factor pioneering and enhancer activation [ChIP-nexus]
GSE218852 Chromatin accessibility in the Drosophila embryo is determined by transcription factor pioneering and enhancer activation
Relations
BioSample SAMN31886376
SRA SRX18393542

Supplementary file Size Download File type/resource
GSM6757746_orer_cad_mbt_nexus_1_negative.bw 52.4 Mb (ftp)(http) BW
GSM6757746_orer_cad_mbt_nexus_1_peaks.narrowPeak.gz 1.2 Mb (ftp)(http) NARROWPEAK
GSM6757746_orer_cad_mbt_nexus_1_positive.bw 52.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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