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| Status |
Public on Jul 02, 2019 |
| Title |
RNA-Seq ClkKO ZT04 rep2 |
| Sample type |
SRA |
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| Source name |
3 brains
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| Organism |
Danaus plexippus |
| Characteristics |
time: ZT04
tissue: Brain
genotype: Clk KO
Sex: female
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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 |
total RNA |
| Extraction protocol |
Brains free of eye photoreceptors were dissected in 0.5X RNA later (Ambion) to prevent RNA degradation, and stored at -80ºC until use. Total RNA was extracted using an RNeasy Mini kit (Qiagen).
PolyA+ RNA was isolated from 2 μg of total RNA with NEBNext Oligo d(T) magnetic beads (New England Biolabs), and multiplexed libraries were prepared using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos (New England Biolabs) and amplified with 12 PCR cycles, following the manufacturer’s recommendations. Libraries quality and size distribution was verified on a Bioanalyzer, libraries were quantified by real-time quantitative PCR, and 16 multiplexed libraries were mixed in equimolar ratios and sequenced on a Hi-seq 2500 (Illumina) using 50bp single end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| 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 2.2.38 and RTA 1.18.61 software with default parameter settings. Sequencer .bcl basecall files were formatted into .fastq files using CASAVA_v1.8.2 script configureBclToFastq.pl.
Reads were mapped to the monarch genome (assembly v3; (Zhan and Reppert 2013)) using TopHat2 (Kim, Pertea et al. 2013) with parameters "--read-realign-edit-dist 2 -g 1 --b2-sensitive”.
Expression levels of transcripts were quantified using Cufflinks (Trapnell, Williams et al. 2010, Trapnell, Roberts et al. 2012).
A modified Fourier transformation was used (Wijnen, Naef et al. 2005) to identify cycling transcripts meeting the following criteria: (1) three or more reads per kilo base per million mapped reads (RPKM) in at least one time point, (2) F24 (24-hr spectral power) > 0.45, (3) p-value of the F24, as described in (Menet, Rodriguez et al. 2012) < 0.05 (i.e., experimental pF24-value is within the top 5% of the 10,000 pF24-values calculated from randomized permutation), and (3) amplitude (maximal/minimal experimental values) > 1.3.
Heat maps were produced using heatmap.2 in gplots package for R. Gene ontology and KEGG pathway enrichment analysis were performed using Metascape (metascape.org).
Genome_build: Monarch genome (assembly v3)
Supplementary_files_format_and_content: Output from cufflinks in generic FPKM Tracking Format saved as plain text files containing the estimated gene-level expression values.
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| Submission date |
Nov 13, 2018 |
| Last update date |
Jul 02, 2019 |
| Contact name |
Christine Merlin |
| E-mail(s) |
[email protected]
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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) |
| GSE122442 |
Genome-wide discovery of daily transcriptome, cis-regulatory elements and transcription factor footprints in the monarch butterfly brain [RNA-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 |
SAMN10413475 |
| SRA |
SRX5001275 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3466647_RNA-Seq_ClkKO_ZT04_rep2_genes_fpkm.txt.gz |
386.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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