GSM2359779: wt 0h rep3 (siRNA-Seq); Schizosaccharomyces pombe; RIP-Seq
Ago1-bound sRNAs were isolated and sequenced using the protocol described in Holoch and Moazed (Holoch and Moazed, 2015b).  Briefly, cell pellets from freshly grown proliferative and quiescent cells were frozen as droplets in liquid nitrogen. Cells were lysed by grinding in Retsch Cryomill (pre-chilled in liquid nitrogen) using Tissue Lyser II (5x 2 min at 30Hz). Lysed cells were thawed at room temperature for 10 min and then resuspended in 1ml of IP buffer (50 mM HEPES, pH 7.4, 5mM Magnesium acetate, 1mM EDTA, 1mM EGTA, 300mM Sodium acetate, 5% glycerol, 0.5% CHAPS, 1 mM PMSF and Roche complete EDTA-free protease-inhibitor cocktail) per gram of lysed cells. The lysate was clarified by centrifugation and the supernatant was incubated with pre-equilibrated anti-DYKDDDDK magnetic microbeads (Medna Bio # P8101ML1) for 4 h at 4 °C. Beads were washed 3x 1 ml lysis buffer, resuspended in 500 μl of nuclease free water, subjected to Phenol/chloroform extraction followed by ethanol precipitation. The RNA samples were resuspended in nuclease free water, electrophoresed on 17.5% Polyacrylamide/7M Urea gel and RNA species between 20-30 nucleotides were gel-extracted. Gel slices were crushed and resuspended in 0.4M sodium acetate (pH = 5.2) overnight at 4 deg C. Extracted siRNAs were ethanol precipitated and resuspended in nuclease-free water. These purified siRNAs were used for library preparation as described previously (Holoch and Moazed, 2015b). Custom library based on Holoch and Moazed, 2015b
GEO Accession
GSM2359779