GSM8561018: Rpp14-GFP_cJL477_rep1_IP; Homo sapiens; RIP-Seq
After dox induction cells were harvested after incubation with 5 mM EDTA in 1X PBS for 5 minutes at 37 °C. Harvested cells were pelleted by centrifugation at 200 x g. Cells were washed once with 1X PBS and then once with 1x lysis buffer (25 mM HEPES pH 8.0, 2 mM MgCl2, 0.1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 300 mM KCl and 10% glycerol). Cells were pelleted between each wash step. Finally, the cells were resuspended in a 1:1 ratio in 1x lysis buffer and flash frozen in liquid nitrogen before being stored at -80 °C. Polysome lysis buffer (20 mM Hepes pH 7.5, 100 mM KCl, 5 mM MgCl2, 1% Triton X-100) supplemented with 1% CHAPS, 1 cOmplete EDTA-free protease inhibitor cocktail (Roche), and 0.02 U/μl of SUPERaseIn (Invitrogen) was added to each frozen cell pellet before thawing the cells in a 37 °C water bath. Cells were lysed by 10 cycles of sonication at high amplitude with a 30 second on time and 1 minute off time (Bioruptor, Diagenode). The lysate was clarified by centrifugation at 21,000 x g for 30 minutes at 4 °C. Clarified lysate was then incubated with Protein A beads (BioRad) coupled to a rabbit anti-GFP antibody at 4 °C for 2 hours on a rotating wheel.  Following incubation with the clarified lysate the beads were washed 4 times for 5 minutes with polysome lysis buffer supplemented with 1 mM DTT, 10 µg/ml leupeptin/pepstatin/chymostatin, and 0.02 U/ml of SUPERaseIn. RNA was eluted from the beads by addition of TRI reagent. For RIP-seq library preparation, libraries were prepared with the Watchmaker library prep kit (Watchmaker Genomics) with a QiaFast Select rRNA kit (Qiagen) module for rRNA depletion and each step was performed according to the manufacturer's instructions.