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Sample GSM5341442

Query DataSets for GSM5341442

Status

Public on May 26, 2021

Title

FAW-25_1

Sample type

SRA

Source name

Spodoptera frugiperda

Organism

Spodoptera frugiperda

Characteristics

strain: The fall armyworm
tissue: Whole body
age: Fourth instar larvae
genotype: Wild type
cell line: No
treatment: 25C

Treatment protocol

To perform transcriptomic analysis fourth instar larvae were incubated at 4 and 40°C for 16 hours as temperature treatment groups to conduct transcriptomic analysis. The control group consisted of larvae incubated at 25°C. A total of 30 larvae were considered for each temperature. The larvae from each group were immediately frozen in liquid nitrogen and preserved at -80°C for subsequent experiments after the temperature procedure.

Growth protocol

The larvae of Spodoptera frugiperda (F0 generation) were obtained from Frotier Agriculture Sciences (Newark, DE, USA). They were raised until pupation under laboratory-controlled conditions (26°C, 70% RH, and a photoperiod of 14 h:10 h [L:D]). The larvae were fed an artificial diet (Newark, DE, USA) during their development. The diet was changed every day for larvae. These larvae were grown in plastic containers with aerated lids measuring 40 x 20 x 15 cm. From the third instar onwards, larvae were reared separately to prevent cannibalism. This was carried out in Petri dishes (8.5 cm diameter). Pupae were sexed and kept with the larvae in the same place. Pupae were monitored on a regular basis before moths appeared.

Extracted molecule

total RNA

Extraction protocol

RNAs samples were extracted from whole body of Sposoptera frugiperda larvae using Trizol reagent (Invitrogen, Carlsbad, CA, USA). After RNA extraction, it was resuspended in nuclease-free water and quantified using a spectrophotometer (NanoDrop, Thermo Scientific, Wilmington, DE, USA). cDNA was then synthesized from RNA (1 μg) using RT PreMix (Intron Biotechnology, Seoul, Korea) containing oligo dT primer according to the manufacturer's instruction.
Illumina sequencing was performed on Macrogen to achieve short-read RNA sequences (Seoul, South Korea). The TruSeQ Stranded MRNA LT Sample Prep Kit (Illumina, San Diego, USA) has been used to build each library with a 1 μg total RNA of the whole group of 5 individuals Spodoptera frugiperda larvae per treatment and with the 101 bp pair end reading HiSeq 400 System (Illumina, San Diego, USA), it was sequenced.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

HiSeq 4000 System (Illumina, San Diego, USA) used for basecalling.
Quality-filtered and adapter-trimmed using Trimmomatic v0.38. FastQC v0.11.7 was used to check data quality before and after trimming. de novo transcriptome assembly was performed using Trinity version trinity rnaseq r20140717, bowtie 1.1.2. Trinity groups transcripts into clusters based on shared sequence content. For assembled genes, longest contigs of the assembled contigs are filtered and clustered into the non-redundant transcripts using CD-HIT version 4.6. ORF prediction for unigenes was performed using TransDecoder version 3.0.1. For the differentially expressed gene analysis, the abundances of unigenes across samples were estimated into read count as an expression measure by RSEM algorithm (RSEM version v1.2.29, bowtie 1.1.2.
Unigenes were searched against Kyoto Encyclopedia of Genes and Genomes (KEGG) v20190104 (http://www.genome.jp/kegg/ko.html), NCBI Nucleotide (NT) v20180116 (https://www.ncbi.nlm.nih.gov/nucleotide/), Pfam v20160316 (https://pfam.xfam.org/), Gene ontology (GO) v20180319 (http://www.geneontology.org/), NCBI non-redundant Protein (NR) v20180503 (https://www.ncbi.nlm.nih.gov/protein/), UniProt v20180116 (http://www.uniprot.org/), and EggNOG (http://eggnogdb.embl.de/) using BLASTN of NCBI BLAST and BLASTX of DIAMOND version 0.9.21 (https://github.com/bbuchfink/diamond) with an E-value default cutoff of E-value 10-5.
Quality check was done for all samples, so that if more than one read count value was 0, it was not included in the analysis. In order to reduce systematic bias, estimates the size factors from the count data and applies Relative Log Expression (RLE) normalization with DESeq2 R library. Using each sample has normalized value, the high expression similarities were grouped together by Hierarchical Clustering Analysis and graphically shown in a 2D plot to show the variability of the total data using Multidimensional Scaling Analysis. Significant unigene results were analyzed as up and down-regulated count by log2FC ≥ 2 and ≤ -2, distribution of expression level between two groups was plotted as volcano plot (https://huygens.science.uva.nl/VolcaNoseR) and simple bar plots. Heat maps were generated using the online tool Heatmapper (http://www.heatmapper.ca/expression/). The KEGG analysis were done to identify the enrichment pathways and heat maps of shared unigenes at both treatment groups (T4 and T40) in comparison with T25 were plotted. In these cases, to get more effective DEGs, the DEGs were limited to log2FC ≥ 5 and ≤ -5, with a P-value<0.01.
The ten genes in response to low (T4) and high temperature (T40) were chosen for validation using RT-qPCR. To do that, FAW larvae were incubated at 4, and 40 °C in separated groups including 10 larvae, for 16 h. RNA extraction and cDNA synthesis were performed according ‘RNA extraction and RT-qPCR’ section. Specific primers were designed using Primer Quest tool (www.idtdna.com) (Table S8). Expression level of EF1α as reference gene was used to normalize target gene expression levels under different treatments. PCR products were assessed by melting curve analysis. Quantitative analysis was performed using comparative CT (2−ΔΔCT) method. Finally, the data compared according ratio of fold change (FC) and ratio of mRNA expression level for all selected genes.
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample ...
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

Submission date

May 25, 2021

Last update date

Sep 28, 2021

Contact name

Mohammad Vatanparast

E-mail(s)

mdvatanparast@gmail.com

Organization name

Animal and Plant Quarantine Agency

Department

Plant quarantine technology center