Oocytes of a single individual of Halocynthia roretzi (Aomori, Japan) were fertilized by sperms of an another single individual and development was followed microscopically. Embryos were snapfrozen in liquid nitrogen at the appropriate stage and kept at -80°C. Total RNA was prepared with the AGPC method and the quality was checked with the Bioanalyzer, most samples had a RIN value of higher than 9. Strand specific libraries were constructed and the sequencing (50PE) was performed with Illumina HiSeq 2000 technology (BGI, Hong Kong, China).

The expression pattern of mRNA encoding CDK9 in Halocynthia egg. CDK9 and PEM mRNA are stained purple and red, respectively. PEM mRNA is shown to indicate the germline cells. Anterior is to the left. Lateral view.

The expression pattern of mRNA encoding PEM in Halocynthia fertilized egg. CDK9 and PEM mRNA are stained purple and red, respectively. PEM mRNA is shown to indicate the germline cells. Anterior is to the left. Lateral view.

The expression pattern of mRNA encoding CDK9 in 4-cell stage Halocynthia embryo. CDK9 and PEM mRNA are stained purple and red, respectively.
Halocynthia CDK9 mRNA was detected in the entire embryo.
PEM mRNA is shown to indicate the germline cells. Anterior is to the left.

The expression pattern of mRNA encoding PEM in 4-cell stage Halocynthia embryo. CDK9 and PEM mRNA are stained purple and red, respectively. PEM mRNA is shown to indicate the germline cells, in the B-line. Anterior is to the left.

The expression pattern of mRNA encoding CDK9 in 8-cell stage Halocynthia embryo. CDK9 and PEM mRNA are stained purple and red, respectively.
Halocynthia CDK9 mRNA was detected in the entire embryo.
PEM mRNA is shown to indicate the germline cells. Anterior is to the left. Lateral view.

The expression pattern of mRNA encoding PEM in 8-cell stage Halocynthia embryo. CDK9 and PEM mRNA are stained purple and red, respectively. PEM mRNA is shown to indicate the B-line (germline cells). Anterior is to the left. Lateral view.

The expression pattern of mRNA encoding CDK9 in 16-cell stage Halocynthia embryo. CDK9 and PEM mRNA are stained purple and red, respectively.
Halocynthia CDK9 mRNA was detected in the entire embryo.
PEM mRNA is shown to indicate the germline cells. Anterior is to the top. Vegetal view.

The expression pattern of mRNA encoding PEM in 16-cell stage Halocynthia embryo. CDK9 and PEM mRNA are stained purple and red, respectively. PEM mRNA is shown to indicate the germline cells : the B5.2 cell pair. Anterior is to the top. Vegetal view.