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Fly gonads were isolated from embryos carrying EGFP-vasa transgene that express GFP only in poll cells by using fluorescence-activated cell sorting (FACS). Total RNA was extracted from the isolated gonads at embryonic stages 13-16 by using RNeasy Mini kit (QIAGEN). Poly(A)+ RNA was purified Oligotex dT30 (TaKaRa) from the total RNA. cDNA was synthesized and amplified by Long-Distance PCR using SMART technology (Clontech) and directionally cloned into pDNR-LIB vector (Clontech) cDNA inserts are flanked by linker sequences 5'-GGCCATTACGGCCGGG-3' (5' linker: SMART IV Oligonucleotide, Clontech) and 5'-polyA-CATGTCGGCCGCCTCGGCC-3' (3' linker: CDSIII/3' PCR Primer, Clontech).
Nucleotide
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