ciHMX overexpression in Ciona intestinalis embryos

We recently showed HMX is expressed in bipolar tail neurons (BTN) in early embryos of Ciona intestinalis. In order to assess the function of the homeobox transcription factor in this cell fate, we used an overexpression strategy. ciHMX was overexpressed in the epidermis, followed by RNAseq of experimental and control embryos. We then looked for differential expression of BTN fate markers, testing if HMX is able to regulate BTN fate determination. Overall design: The full ciHMX ORF was inserted in a reporter construct, under the control of the epiB promoter, which drives expression in the epidermis. An epiB::YFP reporter construct was also used as a control. After in vitro fertilization and dechorionation, we then electroporated parallel batches with either epiB::YFP only (control) or epiB::YFP and epiB::Hmx (Hmx overexpression) constructs. Embryos were grown to the tailbud stage when YFP was visible, identifying transgenic embryos which were then manually selected and processed for RNA extraction. Three full biological replicates were performed on embryo batches derived from different fertilizations, with each biological replicate combining RNA from at least 50 individual embryos. All six RNA samples were sequenced by Illumina HiSeq4000 following polyA selection, yielding approximately 28 million paired end 75bp reads per sample.