RNA-Seq library construction and sequencing

Samples were collected, and immediately frozen in liquid nitrogen and then stored at -80°C until extraction. Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Residual DNA in the total RNA was removed using DNase (Promega, Madison, WI, USA). First-strand cDNA was synthesized using reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Eight cDNA libraries from four developmental stages were processed for RNA-Seq analysis independently using the reagents provided in the Illumina sequencing kit according to the manufacturer’s instructions. Finally, the eight cDNA libraries were sequenced using the Illumina HiSeq 2500 platform at Biomarker Technologies Co., Ltd (Beijing, China).