GS-FLX 454 pyrosequencing for Waterhemp cDNA
Total RNA was isolated from each of the three sets of tissues (seedlings, stressed plants, reproductive growth) using PureLink Plant RNA Reagent (Invitrogen Corp., Carlsbad, CA). Poly A+ mRNA then was isolated from a pool of equal quantities of the three total RNA preparations and used for cDNA synthesis. The cDNAs were then normalized and prepared for 454 pyrosequencing. Steps from mRNA isolation through to pyrosequencing were performed by the W. M. Keck Center for Comparative and Functional Genomics at the University of Illinois. A preliminary titration run was followed with two bulk runs. The first bulk run was dedicated to waterhemp cDNAs whereas in the second run only half the plate was allocated to waterhemp cDNAs.