Heads were dissected from ago2 mutant adult flies (ago2^{414} allele described in Okamura et al. (2004) Genes Dev. 18:1655-1666) as described in Seitz et al. (Curr. Biol. 18:147-151 (2008)).
Biomaterial provider
Megha Ghildiyal
Extracted molecule
other
Extraction protocol
Total RNA was extracted using the mirVana kit (Ambion), then short (18-30 nt) RNAs were gel-purified. 2S rRNA was depleted as described in Seitz et al. (Curr. Biol. 18:147-151 (2008)).
Label
not applicable
Label protocol
not applicable
Hybridization protocol
not applicable
Scan protocol
not applicable
Description
This RNA sample was oxidized before adapter ligation. Protocol for oxidization: Gel-purified 18-30 nt RNAs from 75 micrograms total RNA were incubated in 100 mM NaAcO (pH=5.5) and 33.3 mM freshly-dissolved NaIO4, for 5 minutes at room temperature, then ethanol-precipitated.
Data processing
Small RNA sequences were extracted from the deep-sequencing reads by the following procedure: For n=29 nt, identify all the reads whose 5´ n-mer is immediately followed by the first trinucleotide (CTG) of the 3´ adapter, and whose 5´ n-mer perfectly maps on the Drosophila melanogaster genome (assembly R5.5 from FlyBase). These inserts are removed from the pool, and flagged as "29 nt-long inserts". Decrease n iteratively (n=28 nt, n=27 nt, ...) and repeat the above procedure at each step, until n=18 nt. Pool all 12 sets (29 nt-long inserts, 28 nt-long inserts, ..., 18 nt-long inserts), to generate the list of genome-matching inserts.
Raw data available at SRA under accession SRX000321
