To assess how larvae of different ages vary in their responses to different settlement cues, we induced individual Amphimedon queenslandica larvae with one of three different settlement cues at 1.5, 3, 5, and 8 hours post emergence (hpe) from the adult sponge. The settlement cues were (1) the articulated coralline algae Amphiroa fragilissima, (2) the crustose coralline algae Mesophyllum sp., and (3) the filtered seawater (FSW) negative control. We used CEL-Seq2, an RNA-Sequencing approach (Hashimshony et al., 2016), to generate transcriptome data for a total of 144 individuals (larvae and settled post-larvae) at 2 hours post induction (hpi) to the different settlement cues. Overall design: Transcriptomic data for a total of 144 individual larvae and settled post-larvae of the sponge Amphimedon queenslandica that were exposed to one of three different settlement cues at 1.5, 3, 5 and 8 hours post emergence (hpe). The settlement cues were (1) the articulated coralline algae Amphiroa fragilissima (2) the crustose coralline algae Mesophyllum sp., and (3) the filtered seawater (FSW) negative control. To ensure that no behavioural interactions or conspecific cues would affect larval settlement responses, each larva was induced with a single piece of the inductive cue (~5 mm length A. fragilissima and ~5mm2 for Mesophyllum sp.) in a separate well of 12-well sterile polycarbonate tissue culture plates with 5 ml of 0.22 µm FSW. Settlement plates were placed on a dark surface under a natural day-night regime and were semi-submerged in flowing water from the reef to mimic ambient temperature fluctuations. Swimming larvae and settled post-larvae were sampled for RNA-sequencing at 2 hours post induction (hpi) to a settlement cue (corresponding to larval ages of 3.5, 5, 7 and 10 hpe). Individual larvae (n = 9) were collected for gene expression analyses at 2 hpi to one of three different settlement cues (A. fragilissima, Mesophyllum sp. and the FSW negative control). Post-larvae (n = 7 but one treatment had 6) were sampled at 2 hpi to the coralline algae A. fragilissima and Mesophyllum sp., but only at 5 and 8 hpe as there was insufficient settled post-larvae at all other induction times. All settled post-larvae had attached to a substrate and were not dislodged by agitation (swirling) of the plate. We also collected larvae (n = 9) aged 0.5 hpe that had not been induced with a settlement cue, to act as the developmental controls. Upon collection, larvae and post-larvae were immediately rinsed in a 50:50 sea water:RNAlater wash, and fixed in 100% RNA later (Invitrogen) at 4°C for 24 hours to allow the solution to thoroughly penetrate the tissue before being transferred to -80°C until processing as per manufacturer’s recommendations. RNA was isolated from individual larvae and settled post-larvae using TRIzol (Sigma) (Jindrich et al., 2017) and stored at -80°C in UltraPure™ DNase/RNase-free water (ThermoFisher) until further processing. PolyA libraries were prepared according to the CEL-Seq2 protocol (Hashimshony et al., 2016) and sequenced across three runs on two lanes of Illumina HiSeq2500 on rapid mode using HiSeq Rapid SBS v2 reagents (Illumina) at the Ramaciotti Centre for Genomics (University of New South Wales, NSW, Australia). CEL-Seq2 reads were processed using a publicly available pipeline (https://github.com/yanailab/CEL-Seq-pipeline). Read counts were obtained from demultiplexed reads mapped to A. queenslandica Aqu2.1 gene models (Fernandez-Valverde, Calcino, & Degnan, 2015).