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| Status |
Public on Jul 02, 2019 |
| Title |
ATAC-Seq Control DNA |
| Sample type |
SRA |
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| Source name |
6 brains
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| Organism |
Danaus plexippus |
| Characteristics |
time: control
tissue: Brain
genotype: wild-type
Sex: female and male
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| Treatment protocol |
Adult monarchs were entrained for a minimum of 7 days after eclosion in 15:9 LD cycles at 25ºC.
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| Growth protocol |
Monarch butterflies were raised in the laboratory on semi-artificial diet under 15 hours light, 9 hours dark (LD, 15:9) conditions in Percival incubators at 25ºC and 70% humidity, as previously described (Markert, Zhang et al. 2016). Adult were housed in glassine envelopes in the same lighting and temperature conditions, and were manually fed a 25% honey solution daily.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Brains free of eye photoreceptors were dissected in ice-cold ringer’s solution and resuspended twice in 600 μl of NP-40 lysis buffer (10mM Tris-HCl at pH 7.5, 10mM NaCl, 3 mM MgCl2, and 0.1% NP-40). Crude nuclei were prepared by homogenizing the brains in a 2 ml, ice-cold, Dounce homogenizer with two strokes of a loose-fitting pestle.
Nuclei was directly subjected to transposition by Tn5 transposase for 30 min at 37°C using the Nextera DNA Library Preparation Kit (Illumina), and the tagmented DNA was then purified using a Zymo DNA Clean & ConcentratorTM-5 Kit, all according to a previously published protocol (Buenrostro et al, 2015). To generate a control naked DNA library, 1ng of genomic DNA extracted with phenol/chloroform was also subjected to transposition by Tn5 transposase and the tagmented DNA was purified following the same procedures. Barcoded libraries were PCR amplified, each using a common custom primer and a unique custom Nextera barcoded primer, as in (Buenrostro et al, 2015). For each library, after an initial round of five PCR cycles, the optimal number of PCR cycles to stop amplification prior to saturation was estimated by real-time quantitative PCR. Five to ten additional PCR cycles were then performed bringing the total number to 10 (for control DNA) to 15 cycles, and the libraries were purified using a Qiagen MinElute PCR purification kit. Libraries quality and size distribution was assessed on a Bioanalyzer, libraries were quantified by real-time quantitative PCR and mixed in equimolar ratios before sequencing on a single lane of using 50bp single end reads on an Hi-seq 2500 (Illumina).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Sequence cluster identification, quality prefiltering, base calling and uncertainty assessment were done in real time using Illumina's HCS 2.2.68 and RTA 1.18.66.3 software with default parameter settings. Sequencer .bcl basecall files were demultiplexed and formatted into .fastq files using bcl2fastq 2.17.1.14 script configureBclToFastq.pl.
Adapter sequence was removed using fastx clipper (hannonlab.cshl.edu/fastx_toolkit/) with options "-Q33 -n -v". Clipped reads were mapped to the monarch genome (v3; (Zhan and Reppert 2013)) using Bowtie2 (Langmead and Salzberg 2012) with parameters "--phred33 –local”. Unmapped reads and mapped reads with low mapping quality of less than 10 were discarded after sorting using SAMtools version 0.1.19 (Li, Handsaker et al. 2009). Mapped reads of high quality were adjusted such that those aligned to the + strand and the – strand were shifted by +4 bp and by –5 bp, respectively, as described in (Buenrostro, Wu et al. 2015).
ATAC-seq peaks were called using MACS2 (Zhang, Liu et al. 2008) with parameters "-q 0.01 --nomodel --shift -100 --extsize 200 --keep-dup all", using reads from naked DNA as control. For each genotype and time point, consensus peaks between biological replicates were generated by merging peaks with overlapping start and end coordinates using HOMER (Heinz et al, 2010). Pairwise comparison of the biological replicates were performed by quantifying in each replicate the density of reads contained within consensus peaks using HOMER. To compare replicates from all genotypes and time points to one another, peaks from all libraries were merged in a set of 37,642 consensus peaks, read densities were quantified in each sample, and correlation was determined using the Pearson correlation coefficient (Eisen, Spellman et al. 198. Given that high level of reproducibility was found between biological replicates, ATAC-seq peaks from replicate libraries of WT, Clk KO and DCyc-like mutant at ZT04 and ZT16 were merged by calling peaks on both replicates at the same time using MACS2 with the same parameters as before. The number of peaks called ranged from 13,555 to 23,222 and were used for subsequent analysis.
Locations of ATAC-seq peaks with respect to genomic features were determined for each condition using merged peaks from biological replicates. Although a few number of peaks could not be annotated because they were present in scaffolds containing no annotated genes, majority of the peaks were assigned (in order of priority) as one of the following: (1) promoter-TSS if present within –1kb to +100bp of the transcription start site (TSS), (2) TTS if within -100bp to +1kb of the transcription termination site (TTS), (3) exon if within any exon, (4) intron if within any intron, or (5) intergenic. Differential peak analysis between conditions was performed on sets of merged peaks using HOMER and DESeq2 (Love, Huber et al. 2014) package in R with default FDR cutoff of < 0.05 and log2 fold-change of > 1 and < ‒1. To visualize ATAC-seq peaks, bigWig files normalized to 10 million reads were generated from bam files and visualized using the Integrative Genomics Viewer (IGV; (Robinson et al, 2011; Thorvaldsdóttir et al, 2013)).
Occupied transcription factor (TF) DNA binding sites within open chromatin regions associated to clock-controlled genes were identified in the ATAC-seq peaks of brains of WT ZT04 and WT ZT16 using the Wellington TF footprinting algorithm of pyDNase (Piper, Elze et al. 2013) with a p-value < 10-10 and false discovery rate (FDR) < 0.01. Footprints common to both time points or specific to either one were identified using the intersect command of bedtools (Quinlan and Hall, 2010) with options -wao. Footprints from each group were further analyzed in WT, Clk KO and DCyc-like mutant at ZT04 and ZT16 using the "dnase_to_treeview.py" script in pyDNase to generate CSV files containing Tn5 integration counts from the center of the footprints flanked by 100 bp on either sides. Heat maps were then generated in Java Treeview (Saldanha 2004 using the CSV files as input. To determine the effect of clock disruption on time-of-day dependent footprints, average profiles were generated using a modified dnase_average_profile.py script in pyDNase to obtain raw Tn5 integration counts from positive and negative strands of the 200 bp regions and averaging them for each bp. Data smoothing was applied using 3-bp rolling averages.
Motif analysis
Genome_build: Monarch genome (assembly v3)
Supplementary_files_format_and_content: bigWig files containing the alignment
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| Submission date |
Nov 13, 2018 |
| Last update date |
Jul 02, 2019 |
| Contact name |
Christine Merlin |
| E-mail(s) |
cmerlin@bio.tamu.edu
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| Phone |
(979)862-2457
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| Organization name |
Texas A&M University
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| Department |
Biology
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| Lab |
Biological Sciences Building East Room 102
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| Street address |
3258 TAMU
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| City |
College Station |
| State/province |
TX |
| ZIP/Postal code |
77843-3258 |
| Country |
USA |
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| Platform ID |
GPL25611 |
| Series (2) |
| GSE122445 |
Genome-wide discovery of daily transcriptome, cis-regulatory elements and transcription factor footprints in the monarch butterfly brain [ATAC-Seq] |
| GSE122447 |
Genome-wide discovery of daily transcriptome, cis-regulatory elements and transcription factor footprints in the monarch butterfly brain |
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| Relations |
| BioSample |
SAMN10413456 |
| SRA |
SRX5001249 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3466667_ATAC-Seq_Control_DNA.bw |
92.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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