title: AGO pull downs Drosophila melanogaster S2 cella
design: Small RNA quantification    Total RNA was extracted from D. melanogaster S2-DRSC cells (Hillary Ashe, University of Manchester) using the Ambion miRvana kit according to the manufacturer's instructions. Small RNA library preparation, using the Illumina TruSeq ® Small RNA Kit, and Illumina sequencing were performed by GATC using a HiSeq 2000 machine.   Ago-1 immunoprecipitation and RNA purification    Ago-1 immunoprecipitation was carried out in S2-DRSC cells. Cells were harvested and incubated with 200 µl lysis solution (Chromotek-GFP-Trap® lysis buffer, 0.5 U/µl RNAse inhibitor  (Roche) and 1x Protease inhibitor cocktail (Roche)) for 30 minutes on ice. Lysates were purified by centrifugation at maximum speed for 20 minutes at 4˚C, and then transferred to Tosylactivated Dynabeads® M-280  (Invitrogen) conjugated with 20 mg anti-Ago-1 antibody (ab5070).  Samples were rotated at 4˚C for 2 hours, followed by two washes, proteinase K digestion and RNA isolation as described in [28], with 1U/µl RNAse inhibitor added to the buffers. RNA recovered after ethanol precipitation was used to prepare small RNA libraries with the Illumina TruSeq ® Small RNA Kit according to the manufacturers instructions. RNA qualities were analyzed with TapeStation and libraries were sequenced on the Illumina MiSeq platform generating 50 bp reads (University of Manchester Genomics Core Facility).