Computational protocol: Variation in life history traits and transcriptome associated with adaptation to diet shifts in the ladybird Cryptolaemus montrouzieri

Similar protocols

Protocol publication

[…] Fourth instar larvae (<24 h old) and female adults (~30 days old and fertile) of C. montrouzieri feeding on P. citri or M. japonica were collected for the following transcriptome comparison. For each life stage and diet treatment, two individuals were randomly collected from the above life history experiment. After ~12 h of starvation, the total RNA of these eight individuals was extracted using TRIzon reagent (CWBIO, Beijing, China) according to the protocol of the manufacturer. RNA quality and quantity was determined using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and Bioanalyzer RNA nano chip (Agilent Technologies, Singapore). Only the RNA samples with 260/280 ratio from 1.8 to 2.0, 260/230 ratio from 2.0 to 2.5 and RIN (RNA integrity number) of more than 8.0, were used for sequencing.Approximately 20 μg of total RNA for one individual was used for the construction of libraries using the mRNA-Seq Sample Prep kit (Illumina Inc., San Diego, CA) according to the protocol of the manufacturer. Equal quantities of libraries (approximately 5 ng per sample) with different indices were mixed and stored in a freezer at −80 °C before sequencing. Sequencing was performed in a v3 flowcell on an Illumina HiSeq 2500 sequencer, using the TruSeq Paired-End Cluster Kit v3 (Illumina PE-401–3001) and the TruSeq SBS HS Kit v3 at 200 cycles (Illumina FC-401–3001), generating 2 × 125 bp reads. Image analysis and base calling was done using the HiSeq Control Software version 1.4 and the Off-Line Base Caller v1.9. About 120 million high quality RNA-Seq reads (with a quality score of >20 for each base) were pooled from Illumina sequencing of each of the eight samples and were then assembled into contigs using Trinity []. All sequencing data were deposited in the NCBI Short Read Archive (SRA) under BioProject ID PRJNA304936 (BioSample accession number: LM1: SAMN04311931; LM2: SAMN04311932; LA1: SAMN04309303; LA2: SAMN04311923; AM1: SAMN04311933; AM2: SAMN04311935; AA1: SAMN04311934; AA2: SAMN04311936). We quantified transcript levels in RPKM (reads per kilobase of exon mode per million mapped reads) []. K-Means clustering was performed by the Euclidean distance method and each centroid was the mean of the points in that cluster. Hierarchical clustering of gene expression was performed by the clustergram function in the Matlab Bioinformatics toolbox with default settings. The FPKM method was used to calculate unigene expression [].The unigenes were searched against nr, Swissprot, COG, KOG and Pfam in BlastX with a cut-off E-value of 10e−5. The results of BlastX annotation were uploaded on Blast2GO to generate GO annotations and mapped to the categories of GO database, and also searched against the KEGG pathway []. To investigate which GO terms and KEGG pathways the DEGs participated in, all of the clustered DEGs were mapped back to the GO and KEGG databases. The statistical significances of the GO enrichment were evaluated by the hypergeometric distribution testing:p=1‐∑i=0m−1MiN−Mn−iNnWhere N is the number of unigenes with GO annotation, n is the number of DEGs with GO annotation, M is the number of unigenes with one specific GO annotation and m is the number of differently expressed unigenes with one specific GO annotation []. In the case of statistical significance of the KEGG enrichment, the rich factors were calculated by (DEG number/number of genes annotated by KEGG)/(number of DEG in pathway/number of genes in pathway).To confirm the results of transcriptome comparisons, the qRT-PCRs were performed on randomly selected genes which were differentially expressed (21 DEGs for the four instar larvae and 19 DEGs for the adults). The concentration of each RNA sample was adjusted to 1 mg/ml with nuclease-free water, and ca. 6 μg of total RNA was used as the template to synthesize first-strand cDNA in a 20 μl reaction system using a Superscript III Reverse Transcriptase kit (Invitrogen) following the protocol of manufacturer. The sequences of the specific primer sets are listed in Additional file : Table S9. The β-Actin gene of C. montrouzieri was used as an internal gene. The quantitative real-time PCRs were performed using the SYBR (R) Green I Nucleic A kit (Invitrogen) according to the protocol of the manufacturer. The cycling parameters were 95 °C for 2 min followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s ending with a melting curve analysis (60 °C to 95 °C in increments of 0.5 °C every 5 s) to check for nonspecific product amplification. Relative gene expression was analyzed by the 2-ΔΔCt method []. […]

Pipeline specifications