Oocytes of a single individual of Ciona intestinalis (Roscoff, France) were fertilized by a mixture of sperm of two individuals and development was followed microscopically. Embryos were snapfrozen in liquid nitrogen at the appropriate stage and kept at -80°C. Samples at stage 0, 8, 12, 15, 21, 26 were collected from the same fertilzation, while sample from stage 11 was independently collected from a different individual. Total RNA was prepared with the RNA II kit of Magerey Nagel and the quality was checked with the Bioanalyzer, all 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). Reads were aligned onto C.robusta genome and considered to reflect the expression of the C.robusta orthologous genes.

WT Ciona robusta unfertilized egg probed for KH.C9.853 expression.
KH.C9.853 expression is observed.

Original annotation:
Pem localized signal was detected. The expression in the posterior end/endodermal strand is maternal. B8.12-line cell expression was detected.

Original annotation: No zygotic signal was detected. The expression in the posterior end/endodermal strand is maternal. B8.12-line cell expression was detected.

Aniseed annotation:
Pem localized signal was detected in B3 cell pair. Weak staining is also detected through the whole embryo.

WT Ciona robusta 8-cell stage embryo probed for KH.C9.853 expression (anterior is left and animal pole is top).
KH.C9.853 is expressed in B4.1 blastomer pair.
Original annotation: In situ hybridization showed that KH.C9.853 mRNA, which encodes a protein with four tudor domains, was indeed localized in the posterior pole of the embryo.

Original annotation:
No zygotic signal was detected. The expression in the posterior end/endodermal strand is maternal. B8.12-line cell expression was detected.

Aniseed annotation:
Pem localized signal was detected in B4.1 cell pair. Weak staining is also detected through the whole embryo.

Vegetal view of a WT Ciona robusta 16-cell stage embryo probed for KH.C9.853 expression.
KH.C9.853 is expressed in B5.2 blastomer pair.
Original annotation: In situ hybridization showed that KH.C9.853 mRNA, which encodes a protein with four tudor domains, was indeed localized in the posterior pole of the embryo.

Original annotation:
No zygotic signal was detected. The expression in the posterior end/endodermal strand is maternal. B8.12-line cell expression was detected.

Aniseed annotation:
Pem localized signal was detected in B5.2 cell pair. Weak staining is also detected through the whole embryo.

Vegetal view of a WT Ciona robusta late 32-cell stage embryo probed for KH.C9.853 expression.
KH.C9.853 is expressed in B6.3 blastomer pair.
Original annotation: In situ hybridization showed that KH.C9.853 mRNA, which encodes a protein with four tudor domains, was indeed localized in the posterior pole of the embryo.

Original annotation:
No zygotic signal was detected. The expression in the posterior end/endodermal strand is maternal. B8.12-line cell expression was detected.

Aniseed annotation:
Pem localized signal was detected in B6.3 cell pair. Weak staining is also detected through the whole embryo.

Original annotation:
No zygotic signal was detected. The expression in the posterior end/endodermal strand is maternal. B8.12-line cell expression was detected.

Aniseed annotation:
Pem localized signal was detected in B7.6 cell pair. Staining is also detected in B7.3 and B7.7 cell pairs.

Original annotation:
No zygotic signal was detected. The expression in the posterior end/endodermal strand is maternal. B8.12-line cell expression was detected.

Aniseed annotation:
pem localized signal was detected in B7.6 cell pair. Staining is also detected in B8.5 and B7.7 cell pairs.

Original annotation:
No zygotic signal was detected. The expression in the posterior end/endodermal strand is maternal. B8.12-line cell expression was detected.

Aniseed annotation:
Posterior blastomeres B7.6 cells produce two daughter cells names B8.11 and B8.12 cells (Shirae-Kurabayashi et al., in press). Ci-pem-11 is redistributed into B8.12 cells in addition to B8.11 cells. It seems that there is an expression in the mesenchyme and in the epidermis.

Original annotation:
No zygotic signal was detected. The expression in the posterior end/endodermal strand is maternal. B8.12-line cell expression was detected.

Aniseed annotation:
Posterior blastomeres B7.6 cells produce two daughter cells names B8.11 and B8.12 cells (Shirae-Kurabayashi et al., in press). Ci-pem-11 is redistributed into B8.12 cells in addition to B8.11 cells. According to the others developmental stages, the mesenchyme and the epidermis may be stained.

Original annotation:
Staining is observed in the mesenchyme. The expression in the posterior end/endodermal strand is maternal. B8.12-line cell expression was detected.

Aniseed annotation:
Posterior blastomeres B7.6 cells produce two daughter cells names B8.11 and B8.12 cells (Shirae-Kurabayashi et al., in press). Ci-pem-11 is redistributed into B8.12 cells in addition to B8.11 cells. it seems that there is expression in the epidermis.

Original annotation:
Staining is observed in the mesenchyme. The expression in the posterior end/endodermal strand is maternal. B8.12-line cell expression were detected.

Aniseed annotation:
Posterior blastomeres B7.6 cells produce two daughter cells names B8.11 and B8.12 cells (Shirae-Kurabayashi et al., in press). Ci-pem-11 is redistributed into B8.12 cells in addition to B8.11 cells. Staining is also detected in the epidermis.

Original annotation:
Staining is observed in the mesenchyme. The expression in the posterior end/endodermal strand is maternal. B8.12-line cell expression were detected.

Aniseed annotation:
Posterior blastomeres B7.6 cells produce two daughter cells names B8.11 and B8.12 cells (Shirae-Kurabayashi et al., in press). Ci-pem-11 is redistributed into B8.12 cells in addition to B8.11 cells. Very weak staining is also detected in the epidermis.