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| Status |
Public on Nov 13, 2019 |
| Title |
RNAseq Migrant ZT04 rep1 |
| Sample type |
SRA |
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| Source name |
RNAseq Migrant ZT04_brain
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| Organism |
Danaus plexippus |
| Characteristics |
genotype: wild-type
Sex: female
tissue: 3 brains
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| Treatment protocol |
After capture and eclosion, migrants, LP and SP were entrained for a minimum of 7 days respectively in 11:13, 15:9, and 10:14 LD cycles at 21ºC.
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| Growth protocol |
Wild-caught fall migratory monarch butterflies (Migrant) were housed indoors in glassine envelopes in a Percival incubator under fall-like conditions with light:dark (LD) cycle set to prevailing light conditions (11 h:13 h LD, 0730–1830 Central Standard Time), a constant temperature of 21 °C, and 70% humidity. Meanwhile, laboratory-raised LP and SP monarchs were obtained from eggs layed on potted tropical milkweed plants (Asclepias curassavica) by fertilized wild-type monarch females, which were manually crossed with wild-type males. Both wild-type females and males were previously maintained in 15-hours light: 9-hours dark at 25°C. Eggs were transferred to petri dishes onto milkweed leaves and divided in two groups placed in Percival incubators respectively under 15-hours light: 9-hours dark (long photoperiod; LP) and under 10-hours light: 14-hours dark (short photoperiod; SP), both at constant temperature of 21°C and 70% humidity. Starting at the second instar, larvae were raised individually on semi-artificial diet until pupation. Post-eclosion, adult monarchs were housed in glassine envelopes and were manually fed a 25% honey solution daily until dissection.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Brains of wild-caught migrants and of monarchs raised indoors in LP and SP were dissected in 0.5X RNA later (Invitrogen) to prevent RNA degradation, the retinal pigmented photoreceptor layer was removed, and the brains were stored at -80oC until use. For each seasonal phenotype/photoperiodic condition, three pooled brains were collected in two replicates at ZT1, ZT4, ZT7, ZT10, ZT13, ZT16, ZT19, and ZT22. For each sample, total RNA was extracted using an RNeasy Mini kit (Qiagen).
For samples from wild-caught migrants, polyA+ RNA was isolated from 2 μg of total RNA with NEBNext Poly(A) mRNA Magnetic Isolation Module (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. For samples from monarchs raised in LP and SP, multiplexed libraries were prepared by the Texas A&M AgriLife Genomics and Bioinformatics Facility using polyA+ RNA isolated from 1 μg of total RNA, and multiplexed libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina), 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 et al, 2013) with parameters “--read-realign-edit-dist 2 -g 1 --b2-sensitive”.
Expression levels of transcripts were quantified in each sample using Cufflinks (Trapnell et al, 2010; 2012).
RAIN (Thaben & Westermark, 2014) and MetaCycle (Wu et al., 2016) were used to identify cycling genes meeting the following criteria: (1) three or more reads per kilo base per million (RPKM) in at least one time point, (2) adjusted p-value from RAIN and meta2d of MetaCycle ≤ 0.05, 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 |
Feb 09, 2019 |
| Last update date |
Nov 13, 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 (1) |
| GSE126336 |
Circadian clock genes and the vitamin A pathway regulate seasonal photoperiodic responsiveness in an insect |
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| Relations |
| BioSample |
SAMN10907707 |
| SRA |
SRX5356924 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3596767_RNAseq_Migrant_ZT04_rep1_genes_fpkm.txt.gz |
393.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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