WIld-type PAT from MCIC August 2009
The goal of this experiment was to sequence large numbers of cDNA tags that query the mRNA-cDNA junction.  A detailed description of the protocol is to be found in Wu et al. (submitted).  Briefly, RNA was isolated from 3 week-old Arabidopsis plants that had been grown in soil in a climate-controlled growth room.  cDNA was prepared from total RNA using an anchored primer that contained sequences to permit incorporation, by PCR, of one of the two Illumina adapters required for paired-end sequencing.  Double-stranded cDNA was digested with one of two restriction enzymes (Nla III or Tai I); these enzymes recognize four-base sequences and leave an unpaired four-base end.  Adapters containing sequences to permit the incorporation, again by PCR, of the other Illumina-compatible sequence were ligated to the digested cDNA, that was then purified and amplified using a limited number of PCR cycles.  Amplified tags were submitted for Illumina sequencing.