GSM6533709: dFOXO-null_male_Ago2IP_35d_exp2; Drosophila melanogaster; RNA-Seq
40 whole flies were homogenized in 500 µl of chilled lysis buffer (Kadener, Menet et al. 2009). Lysates were incubated on ice for 10 minutes and centrifuged at 15,000 rpm for 15 minutes at 4 °C. For the Ago1 immunoprecipitation 4 µl of BRD-GP-3 and 4 µl BRD-GP-4 were added to 100 µl of lysate. For the Ago2 immunoprecipitation, the same was done with BRD-GP-5 and BRD-GP-6. Lysates with antibody were incubated with rotation for 1 hour at 4 °C. 50 µl of 25% slurry Pierce protein A/G magnetic agarose (Thermo Fisher) were prepared for each IP. The magnetic agarose was washed in 1 ml of lysis buffer three times before being resuspended in the original volume. 40 µl of slurry were aliquoted into new tubes, supernatant was removed, and the lysates with antibody were added. Lysates and magnetic agarose were incubated overnight with rotation at 4 °C. Agarose was washed five times for 5 minutes in 500 µl of lysis buffer at 4 °C before elution for RNA extraction with Trizol Small RNA libraries were constructed using the NEBNext® Small RNA Library Prep Set for Illumina®(New England Biolabs).