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| Status |
Public on Jun 01, 2023 |
| Title |
S10_M_2 |
| Sample type |
SRA |
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| Source name |
Male_10 C_larval body
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| Organism |
Plutella xylostella |
| Characteristics |
Sex: Male
rearing temparture: 10 C
tissue: larval body
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| Growth protocol |
The laboratory population of P. xylostella was first collected from cabbage field in Gatton, Queenland, Australia in 2014, and it has been maintained with common cabbages (Brassica oleracea var. sugarloaf) at 25 ± 1°C for generations. To examine the reproductive response such as fecundity and fertility to cold stress, P. xylostella were reared at 10°C with fresh and young common cabbage leaves from egg to adult eclosion. The control group P. xylostella were reared at 25°C from egg to adult eclosion. To study the impact of drastic change in ambient temperature on P. xylostella global gene expression, P. xylostella in two treatments were firstly all reared with fresh and young common cabbage leaves at 25 °C from the egg to the early third instar. The early third instar larvae were then placed in incubator set at 10°C (treatment) and 25°C (control) and reared with fresh and young common cabbage leaves until the mid-fourth instars.
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| Extracted molecule |
total RNA |
| Extraction protocol |
To determine P. xylostella gene expression responses to cold stress in male and female larvae, total RNA samples were collected from mid-fourth instar larvae treated with 10C and 25C (control) regime. This included 12 samples includes three biological replicates for each temperature and sex. Each biological replicate consists eight P. xylostella larvae. Total RNA of whole bodies of samples in each replicate was extracted using Qiazol lysis reagent according to manufacture instruction (QIAGEN; Cat No.: 79306). The RNA samples were treated with DNase I for 1 h at 37°C and then their concentrations were measured using a spectrophotometer and integrity was ensured through analysis of RNA on a 1% (w/v) agarose gel. The quality of the RNA samples was examined by using Agilent RNA 6000 pico kit with Agilent 2100 bioanalyzer (Agilent Technologies Inc.). After checking the RNA quality, total RNA samples were submitted to the Novogene Genomics Singapore Pte Ltd for RNA deep sequencing (RNA-seq).
The NEBNext Ultra II RNA library kit (New England Biolabs) was used for RNA-Seq library construction. Fragmented poly A+ RNA samples were converted to cDNA by random primed synthesis using ProtoScript II reverse transcriptase (New England Biolabs). After second strand synthesis, the double-stranded DNAs were treated with T4 DNA polymerase, 5’ phosphorylated and then an adenine residue was added to the 3’ ends of the DNA. Adapters were then ligated to the ends of these target template DNAs. After ligation, the template DNAs were amplified (5-9 cycles) using primers specific to each of the non-complimentary sequences in the adapters. This created a library of DNA templates that have non-homologous 5’ and 3’ ends. A qPCR analysis was performed to determine the template concentration of each library. The PCR-based cDNA libraries were sequenced using Illumina NovaSeq 6000 (PE150) technology with the insert size between 250-300 bp.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The CLC Genomics Workbench version 12.1.1 was used for bioinformatics analyses in this study. RNA-Seq analysis was done by mapping next-generation sequencing reads, distributing and counting the reads across genes and transcripts. The latest assembly of P. xylostella genome (GCF_000330985.1) was used as reference. All libraries were trimmed from sequencing primers, adapter sequences and low-quality reads. Low quality reads (quality score below 0.05) and reads with more than 2 ambiguous nucleotides were discarded. We applied the quality score of 0.05 as cut off for trimming. The Phred quality scores (Q), defined as: Q= -10log10(P), where P is the base-calling error probability, can then be used to calculate the error probabilities, which in turn can be used to set the limit for which bases should be trimmed. Clean reads were subjected to RNA-Seq analysis toolbox for mapping reads to the reference genome with mismatch, insertion and deletion cost of 2, 3 and 3, respectively. Mapping was performed with stringent criteria and allowed a length fraction of 0.8 in mapping parameter, which encounter at least 80% of nucleotides in a read must be aligned to the reference genome. The minimum similarity between the aligned region of the read and the reference sequence was set at 80%.
The relative expression levels were produced as RPKM (Reads Per Kilobase of exon model per Million mapped reads) values, which consider the relative size of the transcripts and only uses the mapped-read datasets to determine relative transcript abundance. To explore genes with differential expression profile between two samples, CLC Genomic Workbench uses multi-factorial statistics based on a negative binomial Generalized Linear Model (GLM). Each gene is modelled by a separate GLM and this approach allows us to fit curves to expression values without assuming that the error on the values is normally distributed. TMM (Trimmed mean of M values) normalization method was applied on all data sets to calculate effective library sizes, which were then used as part of the per-sample normalization (Robinson & Oshlack, 2010). The Wald Test was also used to compare each sample against its control group to test whether a given coefficient is non-zero. We considered genes with more than 2-fold change and false discovery rate (FDR) of less than 0.05 as statistically significantly modulated genes.
All differentially expressed genes were uploaded to Blast2GO server for functional annotation and GO analysis. We used BLAST and InterProScan algorithms to reveal the GO terms of differentially expressed sequences. More abundant terms were computed for each category of molecular function, biological process and cellular components. Blast2GO has integrated the FatiGO package for statistical assessment and this package uses the Fisher’s Exact Test.
Genome_build: GCF_000330985.1
Supplementary_files_format_and_content: TPM, RPKM values
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| Submission date |
Jul 08, 2020 |
| Last update date |
Jun 01, 2023 |
| Contact name |
Kayvan Etebari |
| E-mail(s) |
k.etebari@uq.edu.au
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| Organization name |
The University of Queensland
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| Department |
School of Biological Sciences
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| Lab |
Insect host-pathogen Interaction
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| Street address |
Building No 8
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| City |
St Lucia |
| State/province |
QLD |
| ZIP/Postal code |
4072 |
| Country |
Australia |
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| Platform ID |
GPL28843 |
| Series (1) |
| GSE154004 |
Sex dependent transcriptomic and reproductive response of the diamondback moth (Plutella xylostella) to cold stress |
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| Relations |
| BioSample |
SAMN15473786 |
| SRA |
SRX8686324 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4661641_S10_M_2_GE_.txt.gz |
807.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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