Mud loach skin transcriptome sequencing
cDNA library construction was performed at SinoGenoMax Co., Ltd, Beijing, China (http://www.sinogenomax.com/). Before library construction, the integrity of RNA sample was confirmed using Agilent 2100 Bioanalyzer (with a RNA integrity number of 9). 4 µg of Total RNA was used for isolation of polyA RNA with Sera-mag Magnetic Oligo (dT) Beads (Illumina). The purified mRNA was fragmented into small pieces (100-400 bp) using divalent cations at 94°C for 5 minutes. Double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, Camarillo, CA) with random hexamer primers (Illumina). The synthesized cDNA was subjected to end-repair, phosphorylation, 3’ adenylation and adapter ligation in sequential. After these steps, cDNA fragments at the length of 300 ± 50 bp were selected and purified by gel electrophoresis. Finally, the purified cDNA template was enriched by PCR amplification and the quality of the library was validated in a LightCycler480 (Roche Diagnostics) with an Illumina PhiX174 Control.