RNA-Seq of Pycnopodia helianthoides: adult male tube feet
Total RNA was submitted to Novogene (Sacramento, CA, USA) for quality control, library preparation, and sequencing. RNA integrity and quantitation was assessed on an Agilent 2100. Library preparation consisted of mRNA enrichment using oligo(dT) beads and rRNA removal using the Ribo-Zero kit, then mRNA fragmented randomly with fragmentation buffer. cDNA was synthesized using the mRNA template and random primers of hexamers, followed by second-strand synthesis. After terminal repair, A-ligation and sequencing adapter ligation, the double-stranded cDNA was size-selected and enriched using PCR to generate the final library. A Qubit 2.0 was then used to assess the library concentration, Agilent 2100 to assess the insert size, and qPCR to quantify the library effective concentration. PE150 sequences were generated for the libraries on a NovaSeq 6000 targeting 12Gb of raw data per sample.