RNA-seq of Triops longicaudatus: stage III head
Each sample was placed in liquid nitrogen and homogenized using a mortar and pestle. Total RNA was isolated using RNeasy mini kit (Qiagen, Germantown, MD, USA) following the manufacturers protocols with slight modifications. RNase-free DNase set (Qiagen, Germantown, MD, USA) was used for RNA purification, and total RNA was eluted in 30l of RNase-Free water (Qiagen, Germantown, MD, USA). The A260/A280 ratio, A260/A230 ratio, and concentration of total RNA (>50ng/l) were analyzed with a NanoDrop-2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Eluted RNA was stored in a deep freezer (-80) and then submitted to NICEM (National Instrumentation Center for Environmental Management, Seoul, Republic of Korea) for further processing. To assess the integrity of the total RNA, the samples were examined using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). cDNA library was prepared with NEXTflex Rapid Directional mRNA-Seq kit (PerkinElmer, Waltham, MA, USA) and quality-controlled with Caliper LabChip GX HT DNA High Sensitivity kit (PerkinElmer, Waltham, MA, USA). Finally, raw sequence reads were generated using the Illumina NovaSeq 6000 system (Illumina, Inc., San Diego, CA, USA)