Gs dna library preparation kit

RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA). A Micropoly (A) Purist TM mRNA purification kit (Ambion, Shanghai, China) was used to purify the mRNA under the manufacturer’s instructions. The mRNA from M. enterolobii at mixed stages was treated with DNase I at 37 °C for 25 min to remove any residual DNA. The cDNA was then synthesized using SuperScriptTM III Reverse Transcriptase (Invitrogen) at 42 °C for 1 h. The synthesized cDNA was fragmented into 300–800 bp using an ultrasound sonicator (Fisher, Shanghai, China) and purified using Ampure beads (Agencourt, Suzhou, China). The purified cDNA was prepared with a GS DNA Library Preparation kit (Roche Applied Science, Penzberg, Germany), amplified using the GS emPCR kit (Roche Applied Science) and then sequenced in a Roche 454 Genome Sequencer FLX machine.

Universal primer PCR targeting the 549–567 bp cpn60 UT region was performed using a mixture of cpn60 primers consisting of a 1:3 M ratio of primers H279/H280:H1612/H1613, as described previously [41 (link)–43 (link)]. To allow multiplexing of samples in a single sequencing run, primers were modified at the 5′ end with one of 24 unique decamer multiplexing identification (MID) sequences, as per the manufacturer’s recommendations (Roche, Brandford, CT, USA). Amplicons were pooled in equimolar amounts for sequencing on the Roche GS Junior sequencing platform. The sequencing libraries were prepared using the GS DNA library preparation kit, and emulsion PCR (emPCR) was performed with a GS emPCR kit (Roche Diagnostics, Laval, Canada).
Samples were handled in small batches to avoid cross-contamination, and experimental controls were included at several steps in the study. Regular monitoring of DNA extraction controls in our lab by universal PCR confirms that these procedures are sufficient to eliminate detectable template contamination of study samples. A no template control was also included in each set of PCR reaction as negative controls. Experimental controls were not sequenced as they did not yield any amplification.