Dataset: Gulf of Alaska copepods: annotated transcriptomes
Deployment: TXF17

Sample collection: Zooplankton were collected from depth (2015, 2017, 2018, and 2019) at two stations in Prince William Sound: “PWS2” (Lat: 60°32′. N, Long: -147°48.2′ W) and “PWS3” (Lat: 60°40.0′ N, Long: -147°40.0′ W,) and the Gulf station “GAK1” (Lat: 59º50.7′ N, Long: -149º28′ W). Collection date, station and depth stratum for each individual are given in Hartline et al. (2023) and Roncalli et al. (2019). Zooplankton collections were made using vertical net tows with either a QuadNet with two 150 µm and two 53 µm mesh nets (April and May collections), or a multiple opening and closing plankton net (0.25 m2 cross-sectional area; 150 μm mesh nets; Multinet-Midi, Hydro-Bios; September collections). Zooplankton samples were diluted, and copepods were sorted under a dissection microscope to select individuals from the target species. Briefly, live and undamaged individuals were identified and staged using morphological criteria and preserved in RNALater Stabilization Reagent. Preserved copepods were frozen first in -20ºC during the cruises, and then transferred to −80°C until further processing. Species identification were confirmed through the COI sequence in the assembled transcriptomes.

Total RNA extraction, library construction, RNA sequencing and quality control: For each target species, total RNA was extracted from individuals using QIAGEN RNeasy Plus Mini Kit (catalog # 74134) in combination with a Qiashredder column (catalog # 79654). Selection for sequencing was based on high RNA yields and purity of extraction (RIN>8). The final list included pre-adults (CV) for Neocalanus flemingeri (n=3), Neocalanus cristatus (n=1), Calanus marshallae (n=2), Eucalanus bungii (n=1), an adult male (developmental stage CVI) for Neocalanus plumchrus (n=1) and an adult female for Metridia pacifica (n=1). 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, 150 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. The same workflow was applied to all nine datasets.