GSM5375154: SK-MEL-28 control 40S fraction rep 3; Homo sapiens; OTHER
Cells were treated with 100 µg mL-1 of Cycloheximide (CHX, Sigma-Aldrich) for 12 min at 37 °C and resuspended in lysis buffer.  Lysates were then loaded on a sucrose gradient (the linear sucrose gradient 5-20%). Samples were then centrifugated in an SW41Ti rotor (Beckman Coulter) at 37.000 rpm for 170 min at 4 °C. The fractions were obtained with a BioLogic LP System (Bio-Rad). 14 fractions were collected from each sample, with each fraction having a final volume of 600 µL. From the initial 14 fractions, 4 final samples were obtained (by pulling together some of them): 40S, 60S, 80S and polysomes. Libraries were prepared with TruSeq Stranded Total RNA kit (Illumina) according to the manufacturer's instructions.