GSM4154186: RNA-seq from DENR KO Hela Cell Line 1; Homo sapiens; RNA-Seq
For RNA-seq, cells were lysed with TRIzol and total RNA was isolated following manufacturer's protocol. For Ribo-seq, cells were briefly rinsed with ice-cold PBS containing 10 mM MgCl2, 400 μM cycloheximide (CHX). This solution was poured off, removed by gently tapping the dish onto paper towels, and cells were lysed with 150 µl of lysis buffer (0,25 M HEPES pH 7.5, 50 mM MgCl2, 1 M KCl, 5% NP40, 1000 μM CHX) per plate. Cells were scraped off and lysate was collected. After brief vortexing, lysate was clarified by centrifuging for 10 minutes at 20.000 g at 4C. Approximate RNA concentration was measured using a Nanodrop system and 100U of Ambion RNAse 1 was added per 120 µg of measured RNA. Lysates were incubated with RNAse for 5 minutes on ice.  Lysates were then pipetted onto 17.5-50% sucrose gradients and centrifuged at 35.000 rpm for 3.5 hours in Beckmann SW40 rotor. Gradients were fractionated using a Biocomp Gradient Profiler system and 80S fractions were collected for footprint isolation. RNA was isolated from these fractions using Acid-Phenol extraction and analysed on an Agilent Bioanalyzer system to asses RNA integrity. RNA samples were depleted of ribosomal RNA using the Illumina Ribo-Zero Gold kit. Depleted total RNA was then fragmented using chemical cleavage in 50 mM NaHCO3 at pH 10, 95°C for 12 minutes. Then total RNA was processed in parallel with the depleted RNA from 80S ribosome fractions. For size selection, RNA was run on 15% Urea-Polyacrylamide gels and fragments from 25-35 nt were excised using reference ssRNA nucleotides of 25 and 35 basepairs run on a neighboring lane. RNA was extracted from the gel pieces and phosphorylated using T4 PNK. Deep sequencing libraries were prepared from these RNA fragments using the Bio-Scientific NEXTflex Small RNA-Seq Kit v3. DNA was amplified with 9 PCR cycles for the Ribo-seq samples and 11-13 cycles for the RNA-seq samples. Deep-sequencing libraries were sequenced on the Illumina Next-Seq 550 system.