title: GSM5471997: sR23_tot_siHuR_Doxo; Homo sapiens; RNA-Seq
library_construction_protocol: For preparation of nuclear and cytosolic (protocol 2) fractions, cells were washed twice with ice-cold phosphate buffer saline (PBS) and harvested by scraping. Cells from each plate were pelleted in microfuge tubes, resuspended in 200 μL lysis buffer (10 mM Tris-HCl pH 8,140 mM NaCl, 1.5 mM MgCl2, 10 mM EDTA, 0.5% NP40 and protease inhibitor cocktail) and incubated on ice for 5 min followed by centrifugation at 470 g for 5 min at 4C.The supernatant was stored as cytosolic fraction and the pellet was washed twice gently with lysis buffer before being resuspended in lysis buffer as nuclear fraction. For total cell lysates, cells were lysed directly by pipetting in lysis buffer. The cell lysate fractions were then sonicated (12 cycles, 30:30) on a Diagenode sonicator and used for RNA isolation or western blot. For preparation of polysomal and cytosolic (protocol 1) fractions, cells were incubated at 37 °C with 100 μg/mL cycloheximide in fresh medium for 5 min. Cells were then washed, scraped into ice-cold PBS supplemented with 100 μg/mL cycloheximide, centrifuged at 3000 r.p.m. for 5 min. The cell pellets were resuspended into 400 μL of LSB buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 3 mM MgCl2, 0.5 M sucrose, 1 mM DTT, 100 U/mL RNasin and 100 μg/mL cylcoheximide). After homogenization, 400 μL LSB buffer supplemented with 0.2 % Triton X-100 and 0.25 M sucrose was added. Samples were stayed on ice for 30 min and centrifuged at 12,000 g for 15min at 4 °C. The supernatant was adjusted to 5 M NaCl and 1 M MgCl2. The lysates were then loaded onto a 5-50 % sucrose density gradient and centrifuged in an SW41 Ti rotor (Beckman) at 36,000 rpm for 2 h at 4 °C. Fractions were monitored and collected using a gradient fractionation system (Isco). RNA was extracted from the four heaviest polysomal fractions (pooled). RNA from whole or fractionated cell lysates were extracted with TRIzol Reagent (TRIzol-LS for polysomes; ThermoFischer Scientific) according to the manufacturer's instructions, and 1 µl of GlycoBlue (ThermoFisher Scientific) was added for RNA precipitation. RNA was treated with TURBO DNase I (ThermoFisher Scientific) and quantified using a Nanodrop 2000 spectrophotometer (ThermoFischer Scientific). For sequencing, RNA samples were analyzed using an RNA 2100 Bioanalyzer (Agilent). 3'-seq libraries were prepared with QuantSeq 3' mRNA-Seq Library Prep Kit REV for Illumina (Lexogen) using 250 ng (for polysomal RNA) or 500 ng (for all other samples) of DNase 1 treated RNA (n=3 for each condition) following manufacturer's instructions. Purified libraries were quantified with Quant-iT Picogreen dsDNA kit (ThermoFisher Scientific) and run on Experion automated electrophoresis system (BIO-RAD). Pooled libraries were quantitated by qPCR (KAPA Library Quantification Kits Illumina Platforms, Roche), diluted to 12 pM, and subjected to single-end, 50 bp sequencing using the HiSeq 2500 machine (Illumina).