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| Status |
Public on Aug 05, 2021 |
| Title |
CLAMP-i 0-2hr #3 |
| Sample type |
SRA |
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| Source name |
embryos 0-2hr
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| Organism |
Drosophila melanogaster |
| Characteristics |
developmental stage: embryos 0-2hr
genotype: CLAMP RNAi
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| Growth protocol |
To deplete maternal deposited clamp or zld mRNA throughout oogenesis, we crossed a maternal triple driver (MTD-GAL4, Bloomington, #31777) line with a Transgenic RNAi Project (TRiP) clamp RNAi line (Bloomington, #57008) or a TRiP zld RNAi line (from C. Rushlow lab, Sun et al., 2015). We used the MTD-GAL4 line alone as the control line.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
We conducted ATAC-seq following the protocol from Blythe and Wieschaus (2016). 0-2hr or 2-4hr embryos were collected from grape agar plates, dechorionated by 1min exposure to 6% bleach (Clorox) and then washed 3 times in deionized water. We homogenized 10 embryos and lysed each in 50ul lysis buffer (10mM Tris 7.5, 10mM NaCl, 3mM MgCl2, 0.1% NP-40). We collected nuclei by centrifuging at 500g at 4°C and resuspended nuclei in 5ul TD buffer with 2.5ul Tn5 enzyme (Illumina Tagment DNA TDE1 Enzyme and Buffer Kits). We incubated samples at 37°C for 30min at 800 rpm (Eppendorf Thermomixer) for fragmentation, and then purified with Qiagen Minelute columns before PCR amplification. We amplified libraries by adding 10ul transposed DNA to 25ul NEBNext HiFi 2x PCR mix (New England Biolabs) and 2.5ul of 25uM each of Ad1 and Ad2 primers. We used 13 PCR cycles to amplify samples from 0-2hr embryos and 12 PCR cycles to amplify samples from 2-4hr embryos. Next, we purified libraries with 1.2x Ampure SPRI beads. We performed three biological replicates for each genotype (n=2) and time point (n=2). We measured the concentrations of 12 ATAC-seq libraries by Qubit and determined library quality by Bioanalyzer. We sequenced libraries on an Illumina Hi-seq 4000 sequencer at GeneWiz (South Plainfield, NJ) in 2x150-bp mode.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Demultiplexed reads were trimmed of adapters using TrimGalore (Krueger, 2017) and mapped to the Drosophila genome dm6 using Bowtie2 (v. 2.3.0) with option -X 2000. We used Picard tools (v. 2.9.2) and SAMtools (v.1.9, Li et al., 2009) to remove the reads that are unmapped, not primary alignment, or duplicated (-F 1804), and retain properly paired reads (-f 2) with MAPQ >30. Peak regions for accessible regions were called using HMMRATAC (v1.2.10, Tarbell and Liu, 2019). DiffBind with the DESeq2 method (v. 3.10, Stark and Brown, 2019) was used to identify differentially accessible regions. We used DeepTools (version 3.1.0, Ramírez et al., 2014) and Homer (v 4.11, Givler and Lilienthal, 2005) to generate enrichment heatmaps (CPM normalization), average profiles, motif searches, peak overlap and peak annotation. Visualizations and statistical tests were conducted in R (R Core Team, 2014).
Genome_build: dm6
Supplementary_files_format_and_content: peak: peaks were called using HMMRATAC (v.1.2.10); bigWig files were generated using Deeptools bamCoverage, normalized by CPM.
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| Submission date |
Jun 16, 2020 |
| Last update date |
Aug 05, 2021 |
| Contact name |
Erica Larschan |
| E-mail(s) |
erica_larschan@brown.edu
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| Organization name |
Brown University
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| Department |
MCB
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| Lab |
Larschan Lab
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| Street address |
185 meeting street, Room 368
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| City |
Providence |
| State/province |
Rhode island |
| ZIP/Postal code |
02912 |
| Country |
USA |
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| Platform ID |
GPL21306 |
| Series (2) |
| GSE152596 |
CLAMP and Zelda function together as pioneer transcription factors to promote Drosophila zygotic genome activation [ATAC-Seq] |
| GSE152613 |
CLAMP and Zelda function together as pioneer transcription factors to promote Drosophila zygotic genome activation |
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| Relations |
| BioSample |
SAMN15249773 |
| SRA |
SRX8556251 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4618615_ED-1-3_peaks.narrowPeak.gz |
156.6 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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