454 GS FLX sequencing; Transcriptome of developing testes from the African malaria mosquito Anopheles gambiae
Transcriptome of developing testes from the African malaria mosquito Anopheles gambiae
Testes were dissected under a stereomicroscope using sharpened entomological pins size 000. Care was taken to collect only the gonads with a fragment of vas efferens, but occasionally clumps of fat body cells tightly attached to the apical part of the testis were also taken. In total, testes were dissected from approximately 200 3rd instar and early 4th instar larvae and from 2000 early (up to 4 h old) pupae. An. gambiae G3 strain mosquitoes used in the study were kept in the insectary conditions at 27 °C, 80% RH, and 12h light/12h dark cycle. Larvae were reared in pans with de-ionized water and fed daily with ground fish food (Tetramin). Pans containing fully grown 4th instar larvae were inspected every hour and newly pupated individuals were transferred into clean de-ionized water, and dissected within four hours after collection. Similarly, 3rd and early 4th instar larvae were collected, rinsed in clean water, and used for dissection. Dissected tissues were transferred immediately into RNAlater (Ambion) and stored at -20 °C until RNA isolation. Total RNA was isolated using RNeasy Mini kit (Qiagen) and treated with TURBO DNase (Ambion) according to manufacturers’ protocols.  Then, polyA+ mRNA was isolated using Oligotex mRNA Mini kit (Qiagen) and its integrity evaluated using the Agilent 2100 Bioanalyzer. The mRNA was used to synthesize double-stranded cDNA using the SMARTer™ PCR cDNA synthesis kit (Clontech) according to the manufacturer’s recommendation. Fragmentation of 1 μg of double-stranded cDNA by nebulization, fragment ends polishing, adaptor ligation, isolation and estimation of the single-stranded template library concentration, emulsion PCR with library beads, and loading on the 454 plate was performed according to the published protocol (Margulies M, et al. 2005. Nature 437: 376-380).
Experimental Factor: replicate
run 2
LIBRARY_STRAND
not applicable