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| Status |
Public on Dec 20, 2022 |
| Title |
OreR, GAF, nexus, 2 |
| Sample type |
SRA |
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| Source name |
embryo
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: embryo
genotype: Wild-type (Oregon-R)
developmental stage: 2-3 hours after egg laying
chip antibody: Custom GenScript GAF (aa 1-382)
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| 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.
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| 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.
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| 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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| 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)
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| Submission date |
Nov 27, 2022 |
| Last update date |
Dec 23, 2022 |
| Contact name |
Kaelan Joseph Brennan |
| E-mail(s) |
KBrennan@stowers.org
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| Phone |
8125281836
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| Organization name |
Stowers Institute for Medical Research
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| Lab |
Zeitlinger
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| Street address |
1000 East 50th Street
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| City |
Kansas City |
| State/province |
MO |
| ZIP/Postal code |
64110 |
| Country |
USA |
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| 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 |
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| Relations |
| BioSample |
SAMN31886369 |
| SRA |
SRX18393549 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6757753_orer_gaf_mbt_nexus_3_negative.bw |
22.2 Mb |
(ftp)(http) |
BW |
| GSM6757753_orer_gaf_mbt_nexus_3_peaks.narrowPeak.gz |
892.1 Kb |
(ftp)(http) |
NARROWPEAK |
| GSM6757753_orer_gaf_mbt_nexus_3_positive.bw |
22.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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