library L
The cDNA was  synthesized  using  the  Smart  PCR  cDNA  synthesis  kit  according  to  the manufacturers   suggestions  (Clontech, CA) using 1 mg mRNA. Double-strand cDNA was prepared from 2 mL of the first-strand reaction by PCR (13 cycles). The cDNA was purified using QIAquick PCR purification spin columns (Qiagen) and was checked  for purity and degradation using the Agilent 2100 Bioanalyzer DNA chip.   cDNA normalisation was apllied to the library to decrease the amount of highly abundant transcripts.   1  µg  of  double-stranded  cDNA  was  normalized  using  a  commercial  kit (Trimmer-kit, Evrogen, Moscow, Russia)  that  is  based  on Kamchatka  crab  duplex-specific nuclease. The normalization efficiency was analyzed by quantitative PCR using primers  for Rubisco small subunit (highly redundant) and CP12 (moderately redundant).    Following  normalization,  the  double  stranded  cDNA  was  PCR  amplified,  quality   checked with agarose gels and the Agilent’s Bioanalyzer DNA chip and 3µg of normalized   cDNA  was  used  for  sequencing  with  a  GS  20  or  GS  FLX  sequencer  (Roche/454  Life   Sciences, CT), respectively.