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Sample preparation and sequencing

Daphnid strain and culture conditions
Preparation of polyadenylated RNA
Construction of cDNA libraries
Nucleotide sequencing
Sequence data analysis
Daphnid strain and culture conditions

    The D. magna NIES clone was obtained from the National Institute for Environmental Studies (NIES), Tsukuba, Japan (Tatarazako et al., 2003).
The clone was originally from the Environmental Protection Agency of US (EPA) and has been maintained for more than ten years in NIES in Japan.
Culture medium was prepared by filtering tap water through charcoal and keeping it at room temperature overnight before use.
Cultures of 20 individuals/l were kept in an incubator at 24 1oC with a photoperiod of 14 h light-10 h dark until the daphnids were 3-4 weeks old.
Chlorella were added daily to each culture as a 0.01 mL suspension of 4.3 x 108 cells/mL. The water quality (pH and dissolved oxygen concentration) was measured every two days.
Water hardness, pH, and dissolved oxygen concentrations were 80 mg/L, 7.0 to 7.5, and 80% to 99%, respectively.

Preparation of polyadenylated RNA

    The harvested daphnids were briefly washed and treated with TRIZOL reagent according to the manufacturer's protocol (Invitrogen Corp., Tokyo, Japan) to extract total RNA. Homogenization was performed using the physcotron NS-310E (Nichion, Tokyo, Japan). Poly(A)+ RNA was further purified by employing the Oligotex-dT30 mRNA Purification Kit according to the manufacturer's protocol (Takara Bio., Shiga, Japan).

Construction of cDNA libraries

    The daphnid cDNA library was constructed by using the cDNA Synthesis kit according to the manufacturer's protocol (Stratagene, Tokyo, Japan). For first-strand cDNA synthesis, Superscript II reverse transcriptase (Invitrogen, Tokyo, Japan) was used in presence of 5-methyl dCTP. After second strand synthesis, the EcoRI adaptor was ligated and digested with XhoI. The digested cDNA was then passed though a spin column (Chroma Spin-400, Clontech) to remove linkers and short cDNAs. The recovered fragments were cloned into the EcoRI and XhoI sites of pBluescript IISK(-) and transformed into the E. coli DH10B strain (Invitrogen, Tokyo, Japan) by electroporation.

Nucleotide sequencing

     After transformation, colonies containing Daphnia cDNA were picked and cultured overnight in LB medium (10 g tryptone, 5 g yeast extract, 10 g NaCl per l). Plasmid DNA was prepared using a MultiScreen filter plate (Nihon Milipore Ltd., Tokyo, Japan). Sequence reactions were performed using a DYEnamic ET dye terminator kit (Amersham Biosciences, Tokyo, Japan) followed by sample loading on an automated sequencer MegaBase1000 (Amersham Biosciences, Tokyo, Japan) with the RV-M primer, which can read the nucleotide sequence from the 5' end of each clone.

Sequence data analysis

    The sequence data were analyzed by using the Paracel Clustering Package (Paracel Inc., CA). The nucleotide sequences with phred values higher than 15 and containing at least 300 bp were selected and the vector nucleotide sequences were removed. Phred is a base-calling algorithm based on DNA sequencer trace data that can be an indicator of base-calling reliability. Thereafter, sequences with similarities to ribosomal RNA and mitochondrial DNA were removed. To identify the number of independent EST clones, the EST sequences were clustered by using the Cap3 program and overlapping sequences were grouped into contigs. Each grouped sequence was then translated into amino acid sequences in six frames and subjected to similarity searching against the NCBI non-redundant protein database using the BLASTX program (Altschul et al., 1990). Sequence similarity values were considered to be significant when the expected value was below 1.0E-14 at the amino acid sequence level. It should be noted, however, that the number of groups generated by contig analysis do not necessarily indicate the number of independent genes because non-redundant ESTs may originate from different regions of a single gene.



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