GSM8403804: MUT_Rep3_RPF; Homo sapiens; OTHER
HCT116 cells of the indicated genotype were cultured to 60 to 70% cell density and were washed and scraped gently with ice cold PBS and snap frozen. Cells were then processed for Ribo-seq analysis at the RNA core at Case Western University. The appropriate volume of cell extract containing equal OD was treated with MNase, CaCl2 (5mM final concentration), and Turbo DNAse I and incubated at 25°C for 30 min; the digestion was stopped with SUPERase-inhibitor and placing on ice. The cell extract was then loaded onto a 15%-45% sucrose gradient to separate the polysomes by ultra-centrifugation at 41,000 RPM for 2 hours and 26 min at 4°C. The relevant fractions corresponding to the 80S monosome were collected and precipitated using ethanol and the RNA was isolated by phenol-chloroform extraction. The ribosome-protected fragments were then subjected to denaturing polyacrylamide-mediated gel electrophoresis and were gel-extracted based on size 25nt to 35nt. The RNA was precipitated and subjected to the RiboMinus Ribosomal RNA depletion kit (Ambion). The rRNA-depleted RNA footprints were then subjected to dephosphorylation. Adapter ligation, reverse transcription and subsequent cDNA amplification was performed using the NEXT-Flex smRNA-Seq Kit v3 (Perkin Elmer)