title: GSM8145264: Polysome fractions – Untreated Control – Rep4; Homo sapiens; RNA-Seq
library_construction_protocol: 1 P100 cells per gradient were incubated with 0.1 mg/mL cycloheximide for 10 min on ice to block ribosomes in the elongation step. Then, cells were washed twice with 0.1 mg/mL cycloheximide in cold PBS ad lysed in buffer A (15 mM Tris-HCl pH 7.4, 80 mM KCl, 5 mM MgCl2, 0.1 mg/ml cycloheximide) supplemented with 1% (v/v) Triton X-100, 40 U/ml RNase OUT (Thermo Fisher Scientific), and protease inhibitors (Complete mini, Roche). The lysates were centrifuged 10 min at 14000 g, 4ºC and the clear supernatants were loaded into a linear 10-50% (w/v) sucrose gradient prepared previously in buffer A, and the loaded sucrose gradients were centrifuged at 39000 rpm using a SW40 Ti rotor during 2 h 15 min at 4ºC. The sucrose gradients were fractionated using a fractionation system (ISCO UA-5 UV monitor) measuring the absorbance at A260 to record the polysome profile. Twelve fractions (1 ml) were collected from each gradient. RNA from cytoplasmic lysates and polysomal fractions (8-12) was extracted using phenol-chloroform, isopropanol precipitated, and resuspended in RNase-free H2O. RNA samples were cleaned with the RNeasy kit (Qiagen). RNA quality and concentration was determined using Agilent 2100 Bioanalyzer (Agilent Technologies). cDNA libraries were prepared with 1 µg of RNA using the Illumina TruSeq Stranded mRNA (Macrogen).