Lucilia sericata RNA-Seq
Developmental Profile of the Lucilia sericata Transcriptome
A Lucilia sericata strain was isolated from a Davis, CA population.  Two replicate cohorts of embryos were collected and raised on beef liver.  Samples were flash frozen and stored until RNA isolation.  The following life history stages were considered: embryos (0-6 hours old), feeding third instars (100-101 hours old), postfeeding third instars (117-118 hours old), 0-1 day puparia (165.5-166.5 hours old), 3-4 day puparia (237.5-238.5 hours old), adult females (0-3 days post eclosion), and adult males (0-3 days post eclosion).  Each RNA sample was isolated from a pool of five individuals for each life history stage (except embryos, where an entire mass from multiple females were evaluated) using a Trizol protocol.  Samples from one replicate were used for RNA-Seq experiments on the Illumina platform, while samples from the other replicate were used for subtractive hybridization on the 454 platform.  The embryo samples were multiplexed with other unrelated samples and barcoded with adapters so that each read begins with the sequence CTCT.  The subtractive hybridization involves a pool of driver DNA, which is used to subtract sequences that are shared with the pool of tester DNA.  The following reciprocal subtractions were performed: feeding third instars vs. postfeeding third instars, postfeeding third instars vs. 0-1 day puparia, and 0-1 day puparia vs. 3-4 day puparia.  A Lucilia sericata strain maintained in New Zealand was used to obtain RNA from salivary glands dissected from third instar larvae.  Four independent preparations of salivary gland poly-A+ RNA were made and 454 sequencing was performed using the SMART synthesis kit.
RNA-Seq experiments were performed with Illumina sequencing on the Lucilia sericata transcriptome from the following life history stages: embryos, feeding third instars, postfeeding third instars, 0-1 day puparia, 3-4 day puparia, adult females and adult males.  Two additional libraries were obtained on the 454 platform that include a subtractively normalized library designed to identify differentially expressed genes during the larval and pupal stages and a library from larval salivary glands.  Libraries from Drosophila melanogaster embryos were also obtained to validate our method of calculating de novo RPKM estimates from non-model Lucilia sericata transcripts in the study.