Ciona Genomic DNA and cDNA Amplification

DNA fragments were amplified by PCR with exTaq-HS (Takara Bio) and Phusion HF (New England Biolabs) DNA polymerases from Ciona genomic DNA or cDNA. The primers that we used were summarized in Appendix Table S1. The amplicons were subcloned into TOPO vectors (life technologies). DIG or fluorescein-labeled RNA probes were synthesized by T7 and sp6 RNA polymerases (Roche) from template DNA plasmid digested by NotI or SpeI (New England Biolabs), and were cleaned by RNeasy mini kit (QIAGEN). We followed the protocol for in situ hybridization described before (Christiaen et al, 2009b (link); Ohta and Satou, 2013 (link)). We detected fluorescein and DIG probes using TSA plus (Perkin Elmer), green (FP1168), and red (FP1170), respectively. Primer sequences are provided in Appendix Table S1.
We used an antibody for phospho histone 3 (pH3) that was previously reported (Shirae-Kurabayashi et al, 2006 (link)) (pH3-ser10-6g3-mouse-mAb, Cell Signaling; #9706; 1:500 diluted). The primary antibody was added together with anti-DIG antibody during FISH process, and secondary antibody (anti-mouse-Alexa-555, Thermo Scientific, A-21127) was used to detect the anti-pH3 antibody after detection of ISH probe by using TSA plus (Perkin Elmer) green (FP1168).