34-Xm.3_AL_d0_read_1.fastq.gz
In order to compare gene expression patterns in developing caudal tissue of X.  birchmanni, X. malinche, and their F1 hybrids, we took tissue samples after ten days of regeneration from three pools of ten individual males for each genotype class (90 fish in total) following Offen et al. (37). Samples had to be pooled due to the expectation of low RNA yield from individual samples [Manfred Schartl, personal communication]. Briefly, to begin the experiment we anesthetized each fish in MS-222 and amputated the distal 1/4 of the caudal fin, which includes the sword in X. malinche and F1 hybrids, using a sterile razorblade. After recovery from anesthesia, each fish was housed individually in 11-liter aquaria and fins were allowed to regenerate for ten days at 22C. After ten days, each fish was once again anesthetized and the regenerating blastema was removed. The dissected tissue was divided into three sections, with the most ventral section corresponding to regenerated sword tissue in X. malinche and F1 hybrids. The ventral tissue sections were then pooled in groups of ten according to genotype and replicate for RNA extraction. We generated a total of three pools (30 males) for each biological condition: X. birchmanni, X. malinche and F1 hybrids. RNA was extracted from the pooled tissue using a Trizol based protocol followed by on-column DNAse treatment and purification using the Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA). RNAseq libraries were prepared in a single batch by the Bauer Core at Harvard using the KAPA mRNA HyperPrep Kit (Roche, Palo Alto, CA) with 300-500 nanograms of input RNA. Samples were sequenced across two HiSeq 2000 lanes at Harvards Bauer Core (Data S1D,E)and yielded 150bp paired-end reads.