NextSeq 500 paired end sequencing; ATAC-seq data of Amphimedon queenslandica (Demosponge species) through eight developmental stages, from white stage (cleavage) to adult
Adult Amphimedon queenslandica were collected from Shark Bay, Heron Island, Great Barrier Reef, Queensland Australia (23°26′37.92″ S, 151°55′8.81″ E) under Marine Parks permit number G16/38120.1 Sponges were transferred to a closed aquaria system at the University of Queensland, Brisbane, Australia and maintained as previously described [PMID: 21356734]. Adult brood chambers were dissected and embryos were staged according to [PMID: 17925879]. Larvae naturally released from sponges were collected prior to dissecting brood changes [PMID:21356741].  Cell suspensions from adult sponges were generated by cutting away externally facing tissue, avoiding any potential contaminating tissues and washing three times in 0.2uM filtered Ca+ and Mg+ free artificial seawater (FSW) to try to reduce any potential epiphytes; however, we acknowledge that this tissue may contain allogeneic microorganisms. Cleaned adult tissue was passed through 60um nylon mesh to generate a single cell suspension [PMID: 29942020].Dissected individual embryos were placed in FSW and loaded into a P1000 pipette tip which had been cut and fitted with sealed 60um mesh at the end. This allowed a small volume of FSW to be used to push the embryo through the mesh using a hand-held pipette and the resulting cell suspension to be collected into a 1.7mL centrifuge tube. To collect any tissue stuck on the mesh, the filter tip was then placed in 1 x Trypisn (Sigma)/FSW solution and placed at 37oC for 10mins. This was washed twice with FSW and added to the initial cell suspension. The filter was visually examined under a dissecting microscope to ensure no tissue remained.We first performed the above with larvae and counted the resulting cell suspensions using trypan blue exclusion staining and manual counting with a hemocytometer. From this we estimated a single larvae to have approximately 35,000 cells. Cell suspensions from adult tissues were also counted using trypan blue staining, and an equivalent amount of cells were used to make adult libraries. Cell suspensions of approximately 35,000 cells from individual embryos and adult samples were spin at 500xg for 5mins. ATAC-Seq libraries were made according to [PMID: 25559105, PMID: 24097267]. Libraries using unique primers were amplified for <7 additional cycles. The quality of each individual Library was assessed using a Bioanalyzer High‐Sensitivity DNA Analysis kit (Agilent). Equal volumes of libraries from three individual replicates for each stage were then pooled and run again on the Bioanalyser. Because libraries appeared to have a large concentration of free primer, we incorporated an additional purification step by adding a 1.8x concentration of AMPure Beads (Beckman Coulter), washed twice with 70% ethanol and eluted in 40uL water. A 30nM pooled library was prepared for sequencing.