RNA-Seq of Breviolum minutum SSB01: symbiotic
To analyze SSB01 cells living in hospite, CC7-SSB01 anemones in steady state (6 months after original infection) were collected ~2 d after the last feeding and ~5 h into the light phase. Anemones were homogenized and algal cells enriched from the homogenate using a protocol modified from previous studies 18,26,49 . Briefly, two to five anemones (total wet weight, ~35 mg) were disrupted using a rotor-stator homogenizer for ~10 s in ASW. The homogenate was centrifuged at 3000 x g for 5 min at 4C,and the pellet was washed 3X at 4C with ASW and 1X with ASW containing 0.05% SDS to remove most host tissue. The final pellet was resuspended in ASW, layered onto a 50% Percoll solution in a centrifuge tube, and centrifuged at 9000 x g for 20 min at 4C. The algae did not sediment into the Percoll solution; they were collected, examined microscopically to ensure that they were intact and essentially free of visible host debris, and then pelleted by centrifugation at 500 x g at 4C for 5 min. Total RNA was then extracted from the pellet using the ToTALLY RNA Kit (Ambion AM1910) following the manufacturers instructions for the isolation of RNA from yeast cells; bead beating was necessary for efficient lysis of the algal cells. To constructindexed libraries, ~1 g of total RNA per sample was processed using the TruSeq RNA SamplePrep Kit (Illumina FC-122-1001) following the manufacturers instructions. The resulting libraries were pooled based on their indices (as described in the kit instructions), and end sequencing and clustering of the 101-bp paired-end reads were performed by the Stanford Centerfor Genomics and Personalized Medicine Sequencing Center, using an Illumina HiSeq 2000sequencer.