NextSeq 500 paired end sequencing; Foxh1/nodal defines context-specific direct maternal Wnt/Beta-catenin target gene regulation in early development
Foxh1/nodal defines context-specific direct maternal Wnt/Beta-catenin target gene regulation in early development
For time course RNA-seq analysis on knockdown embryos, biological replicates were generated on a single day by in vitro fertilization using eggs from two independent females.   A total of 22.5ng of foxh1 morpholino was injected into 1-cell stage embryos, with injections distributed over 4 equatorial sites per embryo. For Nodal signaling inhibition, 4-cell stage embryos were immersed in 1/9x MMR containing 100μM SB431542 (Tocris Bioscience) or DMSO as vehicle control. All embryos were cultured at 25°C until desired timepoint/stages. RNA was isolated from ~8-10 embryos per condition per stage as previously described (Chomczynski and Sacchi, 1987), followed by an overnight lithium chloride precipitation. Pellets were washed twice in 70% ethanol and resuspended in DEPC-treated water.  RNA sequencing libraries were generated from full length cDNA prepared using the Smart-seq2 protocol (Picelli et al., 2014), which was then subjected to tagmentation using the Nextera Library Prep kit (Illumina, FC-121-1030).
Experimental Factor: developmental stage
early gastrula stage
Experimental Factor: immunoprecipitate
not applicable
Experimental Factor: protocol
RNA Seq
Experimental Factor: treatment
SB