Solid wildfire system

HEK293T cells were co-transfected with 5 µg Ago2-FLAG plasmid and AgoshRNA-expressing plasmids. Two days post-transfection, cytoplasmic cell extracts were prepared by the treatment of cells on ice for 20 min with IsoB-NP-40 [10 mM Tris-HCl (pH 7.9), 150 mM NaCl, 1.5 mM MgCl₂, 1% NP-40] followed by a centrifugation at 12000 g for 10 min at 4°C. The supernatant was incubated with 75 µl of anti-FLAG M2 agarose beads (Sigma) with constant rotation overnight at 4°C. The beads were washed 3 times in NET-1 buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2.5% Tween 20]. Small RNAs associated with Ago2 were isolated by phenol chloroform extraction followed by DNAse treatment using the TURBO DNA-free kit (Life Technologies). 5 μg RNA was loaded on a denaturing 15% PAGE gel for size fractionation. The 15–55 nt RNA fragments were isolated using a Spin Column (Ambion). The quality of the RNA was assayed on a Bioanalyzer 2100 (Agilent) using a small RNA chip and served as template to create an RNA library that is compatible with the SOLiD sequencing platform. We used the SOLiD Small RNA Library Preparation protocol according to manufacturer's instructions (Applied biosystems; 4452437 Rev. B; page 51 – 66). Samples were run on a SOLiD Wildfire system (Applied biosystems).

293T cells (5 × 10⁶ cells per 25 cm² flask) were co-transfected with 5 μg AGO2-FLAG plasmid (40 (link)) and 20 μg HIV-1 (34 (link)), HIV-rtTA-Tat^wt (35 (link)) or pBluescript plasmid (Stratagene), and cultured for 48 h. HIV-rtTA transfected cells were cultured with 1 μg/ml dox (Sigma; D-9891). AGO2-bound small RNAs were isolated by immunoprecipitation using anti-FLAG M2 affinity gel (Sigma; A2220) as previously described (41 (link)). Five microgram RNA was loaded on a denaturing 15% PAGE gel for size fractionation and the 15–55 nt RNA fragments were isolated using a spin column (Ambion). The quality of the RNA was assayed on a Bioanalyzer 2100 (Agilent) using a small RNA chip. The SOLiD Small RNA Library Preparation protocol (Applied biosystems) was used to prepare an RNA library that was subsequently analysed using the SOLiD Wildfire system (Applied biosystems).