SRP068951 PRJNA309946 GSE77281 RNA-seq based identification of potential RARgamma target genes in Xenopus laevis The development of massively parallel sequencing technologies has revolutionized transcriptome analysis. Sequencing of total cDNA (RNA-Seq) can determine the expression levels of known and novel transcripts with high sensitivity from various developmental stages or conditions. Here we report a robust method for RNA-Seq in Xenopus laevis and apply it to understanding how modulation of retinoic acid signaling alters the transcriptome in early Xenopus laevis development. Three biological conditions were tested: early blastula stage embryos were treated with 1. a retinoic acid receptor (RAR) agonist NRX204647 (4647) at a concentration that stimulated all 3 RAR subtypes (alpha, beta, and gamma); 2. The same agonist at an RAR[gamma] selective dose, and 3. vehicle control. Five single-clutch replicates were obtained for each chemical treatment group, and harvested at neurula stage. We found that single-clutch replicates reflect the stochastic variation of the general outbred population and contribute more statistical power to RNA-Seq experiments due to their feasibility of replication. Our RNA-Seq dataset identified 1590 up-regulated and 685 down-regulated unique genes differentially regulated by all RAR subtypes, and 160 up-regulated and 60 down-regulated genes likely to be regulated specifically by RAR[gamma]. Differential expression detected by RNA-Seq was validated for selected genes by QPCR, which demonstrated nearly 100% quantitative agreement with the deep sequencing data. We further validated RAR-responsive genes by comparison with two previous, published microarray datasets and found substantial agreement. Gene ontology analysis identified RAR targets which may underlie such developmental processes as axial elongation, neurogenesis/synaptogenesis, dorsoventral regulation of the retina, homeotic fate specification, and central nervous system development. We investigated RAR[gamma]-selective targets identified by RNA-Seq and inferred that transcriptional repression by unliganded RARs is of substantial importance to embryonic patterning events. Overall, this paper demonstrates the utility of RNA-Seq in Xenopus laevis, obviating the perceived requirement to use X. tropicalis for genomic analyses. Overall design: Xenopus laevis early blastula stage embyos were exposed to (1) 0.1% EtOH as vehicle, (2) an RARgamma-selective dose (10 nM) NRX204647, and (3) a high dose (1 microM) of NRX204647, which activates all three RAR subtypes (RARalpha, beta, gamma). Each exposure group cosisted of five single-clutch replicates. GSE77281