title: Drosophila melanogaster deep-sequencing small RNAs
status: Public on Apr 11 2008
contact.city: Worcester
contact.first: Hervé
contact.last: Seitz
contact.state: MA
contact.address: 364 Plantation Street
contact.department: Biochemistry and Molecular Pharmacology
contact.country: USA
contact.web_link: http://www.umassmed.edu/bmp/faculty/zamore.cfm?start=0&
contact.institute: University of Massachusetts Medical School
contact.phone: (508)856 1220
organism: Drosophila melanogaster
manufacture_protocol: High-throughput sequencing-by-synthesis (Genome Analyzer, Illumina, San Diego, CA, USA) was as for pyrosequencing in Seitz et al. (Curr. Biol. 18, 147 (2008)) except that RNA Ligase 2 [Rnl2(1-249)K227Q] (Addgene, Cambridge, MA, USA) was used for 3´ ligation. Linkers and primers for sequencing-by-synthesis were: 5´ adaptor, 5´-rGrUrU rCrArG rArGrU rUrCrU rArCrA rGrUrC rCrGrA rCrGrA rUrC-3´ (Dharmacon, Lafayette, CO, USA); 3´ preadenylated linkers, 5´-rAppdCdT dGdTdA dGdGdC dAdCdC dAdTdC dAdAdT ddC-3´.
  After linker addition, the cDNA was synthesized using a reverse transcriptase primer corresponding to the 3´ adapter and amplified by PCR using forward (5´-dAdAdT dGdAdT dAdCdG dGdCdG dAdCdC dAdCdC dGdAdC dAdGdG dTdTdC dAdGdA dGdTdT dCdTdA dCdAdG dTdCdC dGdA -3´) and reverse (5´-dCdAdA dGdCdA dGdAdA dGdAdC dGdGdC dAdTdA dCdGdA dAdTdT dGdAdT dGdGdT dGdCdC dTdAdC dAdG-3´) primers. The PCR pool was gel purified (4% Metaphor Agarose, Cambrex, East Rutherford, NJ, USA) with Qiaex II (Qiagen, Valencia, CA, USA) then sequenced (Genome Analyzer, Illumina) according to the manufacturer’s protocol.