title: Molecular Genetic Studies of Bdelloid Rotifers
design: Total RNA was isolated from clonal cultures of bdelloid and monogonont rotifers using RNAAqueous Micro Kit (Ambion).  Synthesis of cDNA with the first strand primer 5' CTA GAG GCC gAG GCG GCC GAT TTT TTT TTT TTT TTT TTT UVN 3' made use of the template switching property of Superscript II (Invitrogen) to incorporate barcoded biotintylated 5' adapters that matched the "A" sequence priers used in 454 FLX pyrosequencing (5' GCC TCC CTC GCG CCA TCA Gxx xxx GG).   To prepare libraries for pyrosequencing, 3-5 micrograms of cDNA was sheared using an Aeromist Nebulizer (Allied Health products) for 3-4 minutes at 50 psi N2 and the biotintylated 5 EST ens of the fragmented library were capteured with Dynabeads M-280 Streptavidin (Invitrogen).  The captured DNA was end repaired using the Quick Blunk Kit (NEB) and ligated to a modified 454 FLX "B" adapter (AAG CCT TGC CAG CCC GCT CAG T) following A-tailing with Taq polymerase.
library_name: Bm