Klenow fragment 3 5 exo nuclease

To test the effects of the parental environment on seed transcripts, we constructed Illumina Solexa sequencing libraries of 24 self-fertilized dry, ungerminated seeds derived from the same lot of seeds used for the phenotyping. The seeds experienced 1 year of after-ripening before they were used for this experiment. For each genotype and parental environment, we had three biological replicates that consist of a single seed per replicate. For each library, mRNA was isolated from a single seed using Dynabeads mRNA DIRECT Kit from Invitrogen (Product # 610.2, Grand Island, NY) and fragmented using Ambion mRNA Fragmentation Kit (Product #AM8740, Grand Island, NY), followed by cDNA synthesis using random hexamer primers. Double stranded cDNA fragments were blunt-end repaired using Epicentre End Repair (Product #ER81050, Madison, WI) and added a single A to the blunt end using Klenow Fragment 3′-5′ exo-nuclease (NEB Product #M0212L, Ipswich, MA). Ilumina adaptors were ligated to the cDNA fragments using the Epicentre Fast-Link DNA Ligation Kit (Product #LK6201H, Madison, WI). Fragments between 200-400 bp were size selected by agarose gel and samples were indexed and enriched. The libraries were indexed using 12 indices and sequenced on two lanes using the Illumina GAIIx, which generated 76 bp single-end reads.