454 sequencing of Phaseolus coccineus Suspensor Region 2 transcript fragment library
Suspensor regions were micro-dissected from globular-stage embryos six days after pollination from greenhouse-grown plants [Weterings et al., Plant Cell 13, 2409-2425 (2001)]. Double-stranded cDNA was synthesized from suspensor mRNA using the SMART cDNA Library Construction Kit according to the manufacturer (Clontech). The suspensor cDNA fragments were directionally ligated into the SfiI restriction site of the lambda TriplEx2 vector (Clontech), and the recombinant cDNAs were transformed into E. coli XL1-Blue cells (Clontech). Suspensor cDNA plasmids used for directional sequencing were obtained by in vivo excision from the lambda TriplEx2 recombinants in E. coli BM25.8 cells (Clontech).