title: GSM1147067: MOCK; Manihot esculenta; RNA-Seq
library_construction_protocol: Samples from three independent replicates were pooled for RNA isolation and library construction. Control leaves were treated in parallel. Total RNA was isolated from the leaf mixture using the RNAprep pure Plant Kit (Tiangen Biotech; Beijing, China). Six micrograms of total RNA were used for sequencing tag preparation with Digital Gene Expression Sample Prep Kit (Illumina Inc; San Diego, CA, USA) according to the manufacturer's protocol. Using Oligo (dT) magnetic bead, mRNA was purified from each of the two samples, then Oligo (dT) was used as primer to synthesize cDNA. The bead-bound cDNA was subsequently digested with restriction enzyme NlaIII, which recognizes and cuts off the CATG sites. The fragments apart from the 3' cDNA fragments connected to Oligo (dT) beads were washed away and the Illumina adaptor 1 was ligated to the 5' end of the bead-bound cDNA fragments. The junction of Illumina adaptor 1 and CATG site is the recognition site of MmeI, and this enyme could cut at 17bp downstream of the CATG site, and then produce tags with adaptor 1. After removing 3' fragments with magnetic beads precipitation, Illumina adaptor 2 was ligated to the 3' ends of tags, finally acquired tags with different adaptors of both ends formed a tag library. After 15 cycles of linear PCR amplification, 95 bp fragments were purified by 6% TBE PAGE Gel electrophoresis. After denaturation, the single-chain molecules were fixed onto the Illumina Sequencing Chip. Each molecule grew into a single-molecule cluster sequencing template through in situ amplification and was sequenced with the method of sequencing by synthesis (SBS).