GSM2970354: 6h; Bombyx mori; RNA-Seq
Total RNA was extracted from embryos at various times using total RNA isolation kit (Qiagen, 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. The 5' ends of tags were generated by endonuclease NlaⅢ, which recognizes and cut off the CATG sites on cDNAs. The fragments apart from the 3' cDNA fragments connected to Oligo(dT) beads are washed away and the Illumina adaptor 1 is ligated to the sticky 5' end of the digested bead-bound cDNA fragments. The junction of Illumina adaptor 1 and CATG site is the recognition site of MmeI, which is a type of Endonuclease with separated recognition sites and digestion sites. It cuts at 17bp downstream of the CATG site, producing tags with adaptor 1. After removing 3' fragments with magnetic beads precipitation, Illumina adaptor 2 is ligated to the 3' ends of tags, acquiring tags with different adaptors of both ends to form a tag library. The raw tags were sequenced by Illumina HiSeqTM 2000. Clean tags were generated by removing 3' adaptor sequence, low quality tags, tags with a copy number of 1 and tags which were too long or too short, leaving only the tags of 21 bp long.