GSM4932135: HR_RPF_NAT10_rep2; Homo sapiens; OTHER
450 μL of each lysate was treated with 0.3 U/μL RNase I for 40 min at room temperature, while the remaining lysate was flash frozen in liquid nitrogen for later total RNA isolation. Following RNase I digestion (ThermoFisher Scientific, Cat. #: EN0601), 5 μL of Superase-In (ThermoFisher Scientific, Cat. #: AM2694) was added to each sample, and OD260 was measured in the Nanodrop 1000. ~300 µL of lysates was loaded on a 10%–45% sucrose gradient previously prepared in gradient buffer (100 mM Tris-HCl [pH 7.5], 75 mM KCl, 1.5 mM MgCl2) using a BioComp Gradient Master (Short Run, Sucr 10-45% SW41, wt15t). Gradients were spun at 37,000 rpm in a SW 41 Ti rotor for 2 hr at 4°C. Fractions were collected using a Brandel Density Gradient Fractionation system. Ribosome footprinted RNA was precipitated from fractions containing 80S monosomes using 2 volumes of 100% ethanol followed by extraction using TRIzol Reagent. Ribosomal RNA was depleted with the human/mouse/rat Ribo-Zero Gold rRNA removal kit (Illumina). Sequencing libraries were constructed with the Illumina TruSeq Stranded Total RNA Library Prep Kit (RS-122-2201).