Proteinaceous Pheromone Homologs Identified from the Cloacal Gland Transcriptome of a Male Axolotl, Ambystoma mexicanum
Total RNA was extracted from the axolotl cloacal gland tissue using RNeasy Mini Kit reagents (Qiagen, USA) according to manufacturer instructions. Residual DNA was removed using DNase I digestion and cDNA was constructed using the Promega cDNA kit. Briefly, total RNA (1 μg) was reverse-transcribed at 42oC into DNA using 5 U of reverse transcriptase, 1x RT buffer , 1 mM of deoxyribonucleotide triphosphate, 10 ng/μl of random primer, and 20 U RNase inhibitor in a 20 μL reaction. RNA quality and quantity were assessed with an Agilent 2100 BioAnalyzer. Approximately 20 µg of cDNA was used for sequence analysis via Illumina HiSeq™ 2000 platform at the Michigan State University Research Technology Support Facility (East Lansing, MI) to generate 50 bp single-end reads. The raw data from Illumina HiSeq were deposited in the NCBI Short Read Archive (SRA) database.