title: GSM9168205: C18orf21_day3_rep2_total; Homo sapiens; RNA-Seq
library_name: GSM9168205
library_construction_protocol: 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 analysis of knockout cells, cells were washed once with PBS then directly lyzed in 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. For the analysis of rRNA from knockout cells, the total RNA was directly prepped using the Kapa HyperPrep kit without any depletion/enrichment modules according the manufacturer's instructions.  For the analysis of mRNA from knockout cells, the Kapa HyperPrep kit with oligo dT enrichment was performed according the manufacturer's instructions.