GSM6008607: 6h 500 mOsm rep4; Homo sapiens; RNA-Seq
Ribosome profiling: Three 150 mm dishes of pRSV-T cells (80% confluency) were treated with hypertonic stress.  To avoid potential artifacts of pre-treating live cells with cycloheximide, it was added to the lysis buffer only after cells were harvested as previously described (PMID: 26657557, PMID: 30029003, PMID: 25056308, PMID: 31767846). After washing three times with ice-cold PBS, cells were scraped in 1 mL lysis buffer (10 mM HEPES pH 7.4, 5 mM MgCl2, 100 mM KCl, 1% Triton-X,100 μg/mL cycloheximide, 2 mM DTT, RNase inhibitor (1:100 dilution, NEB, M0314L), protease inhibitor (1 tablet per 10 mL, Roche, 04693159001). The lysate was passed four times through a 26G needle and centrifuged at 1300 x g for 10 min. Lysate (OD A260 = 10) was loaded on a linear 10%-50% sucrose gradient (gradient buffer is the same as the lysis buffer, lacking only Triton-X and Protease and RNase inhibitors) and centrifuged at 4°