GSM2701921: CG2; Cloeon viridulum; RNA-Seq
Total RNA was extracted from each sample using TRIZOL Reagent (Cat#15596-018, Life Technologies, Carlsbad, CA, US), following the manufacturer’s instructions, and their RNA integrity was checked in the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US). Qualified total RNA was further purified using the RNeasy micro kit (Cat#74004, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN). Approximately 1 μg of total RNA per sample was used to construct the corresponding cDNA libraries. The library used in RNA-Seq was generated using the Illumina TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). Transcriptome sequencing was carried out by SBC (Shanghai Biotechnology Corporation, Shanghai, China) on the Illumina HiSeq 2500 platform, and approximately 125-bp paired-end raw reads were generated. Reads with low overall quality (proportion of identified bases below 50%), adaptor sequences, ambiguous nucleotides (“Ns”), low quality (quality score below 20), and shorter than 20 nucleotides, and ribosome RNA reads were removed. The remaining clean reads were firstly assembled using CLC Genomics Workbench to obtain the primary unigenes, which were then assembled using CAP3 EST [21] to obtain the final unigenes of real animals, as described for de novo transcriptome assemblies without a reference genome.