RNA-Seq of crinoids
Tissues for RNA sequencing were finely chopped and preserved in RNAlater (Thermo Fisher Scientific) at - 20 C after a 24 fixation at 4 C. The tissue was thawed at room temperature and RNAlater was removed by blotting before proceeding with the next step. RNA was extracted by homogenizing the preserved tissue in Trizol (Thermo Fisher Scientific) with a bead basher for 90 seconds, centrifuging briefly to remove particulates, and then following the manufactures specifications for the Direct-zol RNA extraction kit (Zymo Research). A 10-minute DNAse treatment was performed before storage of purified RNA at -80 C. mRNA libraries were constructed by following manufactures specifications for the KAPA RNA HyperPrep Kits (Roche). Samples were indexed using iTrue style adaptors from Glenn et al. 2016. Transcriptomes were sequenced on two illumina HiSeq Rapid runs (100PE) conducted at the IGM Genomics Center, University of California, San Diego, La Jolla, CA.