RNA-seq of sea urchins
Sequencing libraries were generated from 1.5 g RNA per sample using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following manufacturers protocol with added index codes to attribute sequences to each sample. The library fragments were purified using AMPure XP system (Beckman Coulter, Beverly, USA) to select cDNA fragments in certain length (150200 bp). Polymerase chain reaction (PCR) was implemented with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer, after the process of 3 l USER Enzyme (NEB, USA) and size-selected, adaptor-ligated cDNA at 37 C for 15 min followed by 5 min at 95 C before PCR. PCR products were purified using the AMPure XP system. Library quality was checked using the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was carried out on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) following the protocol.