SRA

Here we employed single cell RNA sequencing to identify the transcriptional program of Nanos and Vasa positive cells and their changes during development. Our single cell sequencing analysis of six developmental stages in P. miniata revealed cell types derived from the three germ layers and expression of the germ cell genes Nanos and Vasa. We used these datasets to parse out 20 cell lineages of the embryo identified by this approach and to focus on the key transitions of germ cell gene expression and test their coexpression with key signaling components. Overall design: Adult Patiria miniata animals were collected by either Peter Halmay (PeterHalmay@gmail.com) or Josh Ross (info@scbiomarine.com) off the Californian coast. Embryos were cultured essentially as described previously (Fresques et al., 2016). Embryos were cultured in filtered (0.2micron) sea water collected at the Marine Biological laboratories in Woods Hole MA, until the appropriate stage for dissociation. All embryos used in the study resulted from mating of one male and one female. Multiple fertilizations were initiated in this study and timed such that the appropriate stages of embryonic development were reached at a common endpoint. The embryos were then collected and washed twice with calcium-free sea water, and then suspended hyalin-extraction media (HEM) for 10-15 minutes, depending on the stage of dissociation. When cells were beginning to dissociate, the embryos were collected and washed in 0.5M NaCl, gently sheared with a pipette, run through a 40micron Nitex mesh, counted on a hemocytometer, and diluted to reach the appropriate concentration for the scRNA-seq protocol. Equal numbers of embryos were used in each time point and at no time were cells or embryos pelleted in a centrifuge (Oulhen et al., 2019).