cDNA synthesis for 454 sequencing

A separate cDNA synthesis step was used for libraries prepared for 454 sequencing and for rtPCR. cDNA was synthesized using the JGI cDNA synthesis protocol version 1.0 (http://my.jgi.doe.gov/general/index.html) with the following modifications: 1) multiple reactions were pooled for phenol:chloroform extractions, 2) an additional chloroform extraction following the phenol:chloroform extraction at step 3 was done, 3) Roche 454 GS20 library preparation specific steps (4–7) were omitted. cDNA was evaluated on a Agilent Bioanalyzer using the DNA 7500 kit (Agilent) with the DNA 7500 assay. The expected, and observed, yield by mass of cDNA from poly-A selected RNA was 50–75%.