GSM9092413: HEK293T_WT_1_HAR; Homo sapiens; OTHER
Cells were scraped, washed with ice-cold PBS, and pelleted by centrifugation at 900 rpm for 10 min. Cell pellets were resuspended in ice-cold PBS and repelleted via centrifugation. Cells are then lysed in freshly prepared lysis buffer A containing 30 mM Tris, pH 7.5, 2% TritonX-100, 20 mM MgCl2, 300 mM NaCl, RNAsin (Promega) and 1X CoMplete Mini protease inhibitor cocktail. Cells were mixed gently by multiple inversions of the culture tube and incubated on ice for 10 min to complete lysis. Cell lysates were cleared of debris and nuclei via centrifugation at 9500 rpm at 4° C for 10 min. The supernatant was loaded on 1.1M sucrose cushion prepared in buffer B (45 mM HEPES KOH, pH 7.5, 150 mM KOAc, 10 mM Mg(OAc)2, and 2mM DTT) and centrifuged for 16 h at 55000 rpm to get the crude ribosomal pellet. Sucrose cushion buffer was treated with bentonite after preparation to inhibit ribonucleases. The pellet was resuspended in Buffer C (20 mM Tris pH 7.5, 150 mM KOAc, 10 mM Mg(OAc)2, RNAsin, and 2mM DTT) to homogeneity, and particles were removed by short centrifugation (10 min at 9500 rpm) and the supernatant was loaded on a linear gradient of 10-40% sucrose. The sucrose buffer was treated with bentonite after preparation to inhibit ribonucleases. The samples were centrifuged at 22,000 rpm for 11 h in a SW-28 rotor, fractions were collected and absorbance (A260) was measured for each. Fractions corresponding 80S peak were pooled together and further centrifuged at 56,000 rpm for 16h at 4˚C. The pellet was suspended in Buffer D [20 mM Tris pH 7.5, 100 mM KOAc, 10 mM Mg(OAc)2, 10 mM NH4OAc and 1 mM DTT], centrifuged for 2.5 h at 75,000 rpm (Ti100 rotor, Beckman). The pellet was dissolved in Buffer D overnight without shaking. The dissolved pellets were flash frozen in aliquots and stored at -800C. Ribo-seq and TIS-seq protocols were adapted from Ingolia, N.T., et al., Nat Protoc, 2012. 7(8): p. 1534-50.