design:  The cDNA library was prepared using a modified template switching protocol (based on the SMART cDNA Library Construction Kit, Clontech) using SuperScript II (Invitrogen 18064-014). 3' and 5' adapters containing SfiI cutting sites were added during first strand synthesis. (5' first strand synthesis primer: (PD242 - AAG CAG TGG TAT CAA CGC AGA GTG GCC ACG AAG GCC rGrGrG, 3' first strand synthesis primer: PD243-30TC - ATT CTA GAG GCC ACC TTG GCC GAC ATG TTT TCT TTT CTT TTT TTT TCT TTT TTT TTT VN). Primary PCR amplification of the library was conducted on a qPCR thermal cycler using primers PCRIIA (AAG CAG TGG TAT CAA CGC AGA GT) and primer PD243-30TC. A control reaction was spiked with 1 x SYBR green I gel stain (Invitrogen S7563) to avoid overcycling. Primary PCR reactions were purified using the Qiaquick purification kit (Qiagen) and cDNA was quantified with Qubit dsDNA BR (MP 32850). The primary PCR product was diluted (1:10) and used in a secondary PCR (10 cycles) to generate the required amount of cDNA for 454 sequencing (>10µg). Secondary PCR product was purified using Qiaquick PCR cleanup kit (Qiagen) and subsequently digested using enzyme Sfi1 (NEB# R0123L) or enzymes Sfi1/Mme1 (NEB#R0637L) in a double digest (see below). Products were size selected using Chromaspin TE-400 columns (Clontech#636076), blunted using NEB kit (NEB# E1201L), and quantified. The sequencing library was prepared using 454 GS FLX Titanium chemistry at http://engencore.sc.edu/.