Dataset: Neocalanus flemingeri incubation experiment: gene expression
Deployment: Lenz_Gulf_of_Alaska_2019-04-15

Zooplankton were collected on a day-trip to station GAK1 (59º50.7′ N, Long: 149º28′ W, depth 264 m, Gulf of Alaska) (http://research.cfos.uaf.edu/gak1/) aboard the M/V Dora on April 15, 2019. Collections were made using QuadNet with two 150 µm and two 53 µm mesh nets towed vertically from 100 to 0 m. Collection details are provided in Roncalli et al. (2023). Zooplankton samples were diluted, brought back to the laboratory and sorted under a dissection microscope to select stage CIV Neocalanus flemingeri individuals. As individuals molted into CVs, they were removed from the holding containers and transferred into 750 ml Falcon flasks with 3 individuals per flask and assigned to one of the 4 food treatments, as described in detail in Roncalli et al. (2023). Three individuals were preserved upon molting (Wk0). Individuals were harvested at 3 incubation times (Wk1, Wk2 and Wk3) and preserved in RNALater Stabilization Reagent. Preserved copepods were frozen first in -40ºC during the experiment, and then transferred to −80°C until further processing. 

Total RNA extraction, library construction, RNA sequencing and quality control: total RNA was extracted from individuals using QIAGEN RNeasy Plus Mini Kit (catalog # 74134) in combination with a Qiashredder column (catalog # 79654). Sequencing was performed on 3 Wk0 individuals and 3 replicate individuals for each time x treatment combination. Total RNA was shipped on dry ice to the Georgia Genomics Bioinformatics Core (https://dna.uga.edu) for RNA-Seq. There, double-stranded cDNA libraries (KAPA Stranded mRNA-Seq Kit, with KAPA mRNA Capture Beads (cat #KK8421]) from each individual were multiplexed and sequenced using an Illumina Next-Seq 500 instrument (High-Output Flow Cell, 75 bp, paired end). Quality of each RNA-Seq library was reviewed with the FastQC software28. From each RNA-Seq library, low quality reads were removed using FASTQ Toolkit (v. 2.2.5 within BaseSpace). Illumina adaptors, reads <50 bp long, reads with an average Phred score <30 and the first 12 bp from each read, were removed from each library.