title: 454 sequencing of Manduca sexta transcript fragment library
design:                The RNA sample is a complex mixture :               Each of day 2, 5th instar Manduca sexta larvae (20) was injected with a                mixture of formaldehyde-killed Escherichia coli (2^107 cells),                Micrococcus luteus (20 micrograms) and curdlan (20 micrograms) in 30 microlitres H2O. Total RNA                samples were isolated from the hemocytes and fat body 24 h later using                TRIZOL Reagent (Invitrogen Life Technology). Hemocyte and fat body total                RNA samples were also prepared from day 3, 5th instar naive (40) and                bar-stage wandering larvae (20). Similarly, integuments and trachea of                the 5th instar naive larvae were dissected for total RNA isolation. The                RNA samples (A260/A280 > 1.8) were combined at the following                percentages: hemocytes (10%), fat body (15%), integument (5%) and                trachea (5%) from naive larvae, hemocytes (20%) and fat body (35%) from                injected larvae, hemocytes (5%) and fat body (5%) from wandering larvae.                mRNA was purified from the pooled total RNA (500 micrograms) by binding to                oligo(dT) cellulose twice (Poly(A)Purist, Ambion).               No vector was used since cloning was not involved, random pentadecamers                were used as primers for 1st and 2nd strand synth               esis.             