Transcriptome Analysis of the Zebrafish Mind Bomb Mutant

To identify the genes differentially expressed in the zebrafish mibta52b mutant, genome-wide transcriptome analysis was performed using Serial Analysis of Gene Expression (SAGE). 335 transcripts were identified whose expressions were significantly altered in the mibta52b mutant as compared with the wild-type. After mRNA isolation using oligo(dT)25 beads (Dynal Biotech ASA, Oslo, Norway), cDNA synthesis was carried out using anchored oligo(dT) primers to reduce truncated cDNA through internal poly(A) priming (Nam et al., 2002). SAGE analysis was carried out as described (Velculescu et al., 1995) but was modified slightly according to Lee et al (Lee et al., 2007). We performed a low-cycle PCR (14 cycles) using the GeneAmp PCR System 2400 (Applied Biosystem, Foster City, CA) to amplify 3’cDNA in order to generate a sufficient amount of templates for SAGE analysis. The sense primer used was a SAGE primer 1 (5’-GGA TTT GCT GGT GCA GTA CA-3’) or SAGE primer 2 (5’-CTG CTC GAA TTC AAG CTT CT-3’); the antisense primer used was 5’-biotin- ACT ATC TAG AGC GGC CGC TT-3’, which was located in the 3’ end of all cDNAs generated from the anchored oligo(dT) primers. BsmFI-released fragments containing SAGE tags, were gel purified before being used for ditag formation and concatenation to provide high-quality tags for SAGE analysis. SAGE tag sequences were collected with Big-Dye sequencing and an ABI3730 sequencer, and tag sequences were extracted with SAGE 300 software. A total of 92,970 SAGE tags were collected from the two libraries.