ITS2 analysis of different cladobranch species
The ribosomal ITS2 region of Symbiociniaceae was amplified using primers targeting the 5.8S and 23S regions: ITS-Dino-fwd and ITS2-Dino-rev2. PCR was performed in a 20 L reaction containing 5 L template cDNA, 10 L DreamTaq Green PCR Master Mix (Thermo Fisher, USA), and 2 L of each primer (10 M). Cycling conditions included 30 cycles of 94C for 1 min, 54C for 1 min, and 72C for 1 min, with an initial denaturation (95C, 15 min) and final extension (72C, 10 min). PCR products were purified with Agencourt AMPure XP beads and indexed via a second PCR (8 cycles, 52C annealing) using Illumina TruSeq adapters. Final products were purified, quantified with a Qubit 4 Fluorometer, pooled equimolarly, and sequenced on an Illumina MiSeq platform (2250 bp paired-end) at Saarland University.