GSM5114406: F50 N2; Bombyx mori; RNA-Seq
Total RNA was extracted from PGs using TRIzol reagent (Invitrogen, New York, NY, USA) in accordance with the manufacturer's instructions. The mRNA was purified by Oligo(dT) magnetic beads (Invitrogen, USA), then used as templates for cDNA synthesis. Poly (A)-tailed RNA prepared using magnetic oligo (dT) beads was broken into short fragments using a fragment buffer and was then reverse-transcribed to synthesize the first-strand cDNA with a random primer, and then DNA polymerase I was mixed with RNase H, dNTP, and the buffer solution to synthesize the complementary strand. The libraries were constructed using the Illumina methods and protocols, following the manufacturer's instructions. The insert size and concentration of the cDNA library were both checked and quantified by an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA) and Qubit® RNA Assay Kit (Life Technologies, CA, USA), respectively. RNA-seq was carried out with an Illumina HiSeq 2500 instrument (Illumina, San Diego, CA, USA). In order to ensure the quality of information analysis, the raw reads were cleaned by removing the adapter sequences, empty reads, unknown nucleotides (ratio ≥10%), and low-quality reads with a basic mass value of Q ≤ 20 which accounted for more than 50% of the whole read length.