Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: For RNA isolation, the pooled daphnids were placed in a new 2-ml Eppendorf tube containing 600 μl of RLT Buffer, β-mercaptoethanol and one 5-mm stainless steel bead. The daphnids were then homogenized in a tissue lyser for 2 minutes. RNA isolation from pooled D. magna samples was then performed with Qiagen RNA mini kits (Product ID# 74104, Qiagen, Germantown, MD, USA). The RNA isolation procedure followed the instructions in the mini kit and included a step treating the samples with DNase solution. The DNase solution preparation followed the kit handbook instructions. Following RNA isolation, RNA concentrations and purity were determined using a Nanodrop 1000 Spectrophotometer (Thermo Fisher Model ND-1000, Thermo Fisher Scientific Inc., USA), and a qualitative RNA integrity examination was conducted using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries were prepared with the TruSeq Stranded mRNA LT Sample Preparation Kit (Illumina, San Diego, CA, USA) as per the manufacturer's instructions using 125 ng of total RNA per sample. Briefly, the poly-A containing mRNA molecules are purified using magnetic beads, fragmented, and synthesized into first strand cDNA. Next, second strand cDNA is synthesized, a single 'A' nucleotide is added to the 3' ends, the single-index adapters are ligated, and DNA fragments are enriched to prepare the final libraries. The size and purity of the libraries were determined on the D1000 ScreenTape on the Agilent TapeStation 2200 (Agilent Technologies, Santa Clara, CA, USA). The quantity of the individual libraries was assessed using the KAPA Library Quantification Kit for Illumina Libraries (Kapa Biosystems, Inc., Wilmington, MA, USA) and confirmed using the dsDNA HS Kit on the Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). The concentrations of the libraries were then normalized. The libraries were pooled together with eight libraries per pool, and the pools were quantified using the dsDNA HS Kit on the Qubit 3.0 Fluorometer, followed by further dilution to 5nM. The pools were then sequenced on the HiSeq 4000 system (Illumina) at 1x150 cycles single-read using the HiSeq 3000/4000 SBS kit following the manufacturer's instructions.