title: GSM2445922: Pj_L3s_egg_mRNA; Parabronema skrjabini; RNA-Seq
library_construction_protocol: Total RNA was extracted from eggs, infective larvae, adult male and female using the TRIzol reagent. RQ1 DNase was added to remove the DNA templates. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260 nm/280 nm (A260/A280) using smart spec plus. RNA integrity was further verified by 1.5% Agarose gel electrophoresis. For each stage, 5μg of total RNA was used for RNA-seq library preparation with the Balancer NGS Library Preparation Kit (Gnomegen). Dyna oligo (dT) beads (Ambion) were used to isolated poly(A) mRNA from the total RNA samples. Purified mRNAs were fragmented into short pieces in fragmentation buffer. RNA fragments were end-repaired and ligated with 5'adaptor. Then reverse transcription was performed using RT primer harboring 3' adaptor sequence and randomized hexamer. The cDNAs were purified and amplified. The PCR products corresponding to 300-500 bps were purified, quantified and stored at -80 ℃ until used for sequencing. For high-throughput sequencing, the libraries were prepared following the manufacturer’s instructions and applied to Illumina Nextseq 500 system for 150nt and Hiseq 2000 system for 90nt paired-end sequencing by ABlife. Inc (Wuhan, China).