Instrument: Illumina Genome Analyzer II
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Three biological replicates were collected for each sample and one replicate of wild-type (AB strain) embryos at the same developmental stage was collected. Each replicate contained pools of 10 whole embryos. Samples were collected in RNAlater (Qiagen, Hilden, Germany) and stored at 4°C until processing. The RNeasy mini RNA extraction kit was used to isolate RNA, on-column DNaseI digestion was performed and RNA was collected in RNase-free water, as per manufacturer’s instructions (Qiagen, Hilden, Germany). A Nanodrop spectrophotometer (Beckman, USA) was used to determine the concentration of RNA and a Bioanalyser (Agilent, Santa Clara, USA) was used to confirm the RNA Integrity Number (RIN) of the samples as 8 or higher. Using 1 μg of total RNA, cDNA libraries were prepared using the mRNA-seq 8-Sample Prep Kit as per manufacturer’s instructions (Illumina, RS-100-0801). Briefly, poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Following purification, mRNA was fragmented and cDNA generated with ligated adaptors. These products were purified and PCR enriched to create the final cDNA library.