Supplementary information on experimental procedures
RNA extraction and cDNA synthesis
Total RNA was extracted from approximately 100 ml of 2 week-old culture using Trizol
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(Life Technologies, Carlsland, CA, USA). Genomic DNA contaminated in the extracted
RNA sample was digested by deoxyribonuclease (DNase) I (Life Technologies) at 65°C
for 10 min. Complementary DNA (cDNA) was synthesized from the DNase-treated
RNA by SuperScript II (Life Technologies) using random hexamers or by 3’RACE
system (Life Technologies). Aliquots of the non-DNase-treated RNA and DNase-treated
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RNA were kept for using as PCR templates (see below).
RT-PCR and sequencing of ubc12 and usp39
The cDNA sample synthesized using 3’RACE system was used as a template for PCR
amplification of ubc12 and usp39. The sequences of primers for ubc12 amplification
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were ubc12 Fw (5’-gcg caa gag caa aag cag caa gag- 3’) and AUAP (5’-ggc cac gcg tcg
act agt ac- 3’), and those for usp39 amplification were usp39 Fw (5’-cga tcc gcc ata agt
1
gtc cg- 3’) and AUAP. The thermal cycling consisted of 45 cycles of denaturing at 94°C
for 30 s, annealing at 55°C for 1 min and extension at 72°C for 2 min. Amplified DNA
fragments were cloned into the pCR2.1 vector with a TOPO TA Cloning Kit (Life
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Technologies). The DNA fragments inserted into the vector were sequenced on both
strands.
Transcription analyses of rRNA genes
A combination of the types of PCR templates (i.e., non-DNase-treated RNA,
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DNase-treated RNA or cDNA synthesized by random hexamers) and the primer sets
(i.e., primers for stramenopile-type 18S rRNA gene, for alveolate-type 18S rRNA gene
or for its adjacent 28S rRNA gene) are summarized in Figure 2c. The thermal cycling
consisted of 45 cycles of denaturing at 94°C for 30 s, annealing at 55°C for 1 min and
extension at 72°C for 1 min. All three rRNA genes analyzed were amplified from the
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non-DNase-treated RNA (lane 1-3 in Figure 2c) and they were not amplified from the
DNase-treated RNA (lane 4-6 in Figure 2c), suggesting that the rRNA gene sequences
in lane 1-3 were amplified from the Genomic DNA contaminated in the extracted RNA
2
and that cDNA synthesized from the DNase-treated RNA is not contaminated with the
genomic DNA. We also sequenced the rRNA gene sequences amplified from the
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non-DNase-treated RNA and confirmed that the specific primers designed for each gene
properly work for PCR amplification (data not shown).
Approximately unbiased (AU) test for alternative positions of UBC12 of Ciliophrys
infusionum
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The branch of C. infusionum was moved to 8 alternative positions in the ML tree. Site
InL data were calculated under the same model as the ML analysis by RAxML. These
lnL data were subsequently analysed by CONSEL v. 0.1 (Shimodaira & Hasegawa,
2001).
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Reference
Shimodaira H, Hasegawa M. (2001) CONSEL: for assessing the confidence of
phylogenetic tree selection. Bioinformatics 17:1246–1247.
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