RNA-Seq of Pseudosphyrapus quintolongus
Sample preservation, RNA extraction, sequencing and assembly were conducted based on methods previously described for spiders (Kono, et al. 2016), with some modifications. Briefly, a single specimen of each tanaidacean was preserved in RNAlater Stabilization Solution, then stored at -80 C. RNA was extracted using a Direct-zol RNA Microprep Kit (Zymo Research). The Illumina library was prepared using the KAPA RNA HyperPrep Kit (KAPA Biosystems) targeting 300 bp fragments, but in order to amplify larger silk-related transcripts, PCR extension time was increased to 3 min per cycle. Extracted RNA was below minimum requirement for KAPA RNA HyperPrep Kit for samples R05 Apseudomorpha sp., R06 Paradoxapseudes littoralis, and R24 Heterotanoides sp., and therefore these samples were prepared using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clonetech) also with increased PCR extension time, followed by KAPA HyperPlus Kit (KAPA Biosystems) fragmentation and cDNA library preparation. The sequence library was then sequenced on a NextSeq 500 (Illumina) with 300 cycles of high-output mode as paired-end reads. Sequences were base called and demultiplexed, and adaptor sequences were removed with bcls2fastq v.2 software (Illumina).