design: To determine changes in DNA and RNA sequences and abundance following stimulation of an immune response, we performed treatments with either lipopolysaccharides (LPS) from Pseudomonas aeruginosa (Sigma #L9143) or cyclic dinucleotide 23-cGAMP (Invivogen #tlrl-nacga23-02) in M-medium. LPS treatments were conducted in dishes containing five sponges 7-days after gemmule plating (stage 3), by replacing M-medium with premixed 5g/L LPS. This concentration was chosen based on unpublished results indicating it induces a significant immune response in freshwater sponges (Scott Nichols, pers. comm.). Juvenile sponges were then harvested for DNA and RNA either one day later (day 8, stage 4) or had their LPS treatment terminated at day 8 by replacing LPS M-medium with fresh M-medium, followed by harvesting at day 12 (stage 5). Treatment with cyclic dinucleotide was performed for four hours on 8-day old (stage 4) and 12-day old (stage 5) sponges by replacing M-medium with premixed 100uM 2,3-cGAMP in M-medium. DNA and RNA were isolated following similar protocols as for our developmental time course samples, resulting in high-quality DNA from two replicates and RNA from one replicate for each treatment and time point.