Hiseq 4000 sequencing platform

Approximately 1000 of adult nematodes were harvested and total RNA was extracted using RNeasy Micro kits from Qiagen. Extracted RNA was thereafter sent to NovogeneAIT Genomics Singapore for library prep and sequencing using Illumina HiSeq4000 sequencing platform (Illumina) in a paired end read approach at a read length of 150 nucleotides. The RNAseq reads from each sample were mapped to the reference C. elegans transcriptome (WBcel235) with kallisto (v0.46.0)^{77 (link)}. The estimated counts were imported from kallisto to the R environment (v3.6) and summarized to gene-level in length scaled TPM units using the tximport package (v1.12.3)^{78 (link)}. The DESeq2 package (v1.24.0)⁷⁹ was used to identify differentially expressed genes (DEGs) while the mixOmics package (6.8.0)^{80 (link)} was used to obtain PCA plot and heatmap.

TRIzol reagent (Invitrogen; USA) was used to isolate total RNA from 18 samples (five groups) according to the manufacturer's instructions. RNA purity and concentration were determined by ultraviolet absorbance using a Nanodrop Nano Photometer spectrophotometer (IMPLEN, USA), while RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Total RNA (3 μg) was used to construct a cDNA library using an RNA Library Prep Kit for Illumina (New England Biolabs, USA), according to the instructions provided by the manufacturer. In brief, total RNA was treated with DNaseI (Invitrogen, Life Technologies, USA) to remove genomic DNA contamination. This was followed by enrichment of poly(A) mRNA using oligo d(T) magnetic beads (Invitrogen). mRNA was next treated with fragmentation buffer, and cleaved RNA was copied into first-strand cDNA fragments using reverse transcriptase (Invitrogen) and random hexamer primers. Second-strand cDNA fragments were synthesized using Second Strand Synthesis Enzyme Mix, including DNA polymerase I, buffer, deoxyribonucleoside triphosphate, and RNase H. After purification and paired-end repair, cDNA fragments were ligated to sequencing adapters and amplified by PCR to obtain the final paired-end library. Libraries were subsequently sequenced using the Illumina HiSeq 4000 sequencing platform (Novogene, Shanghai, China).

After grinding into powder in liquid nitrogen, ovules from each sample were applied to total RNA extraction, followed by isolating mRNAs. The individual cDNA was synthesized using the random hexamers as primers and mRNA templates. The resultant products were connected with adapters, followed by size selection and PCR amplification. The constructed library was analyzed by Illumina HiSeq™ 4000 sequencing platform (BGI, Shenzhen, China). The data of RNA-Seq have been assigned with the accession number SRP158368 in NCBI (http://www.ncbi.nlm.nih.gov/sra).