Instrument: Illumina HiSeq 2000
Strategy: RIP-Seq
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Cycloheximide (50 µg/ml) was added to each pooled 80S fraction (1 ml) obtained by sucrose gradient fractionation. Aliquots of these input fractions were used for Western blot analysis (20 µl) and total RNA extraction with Trizol (50 µl). The rest of the sample was subjected to anti-FLAG immunoprecipitation. To prepare anti-FLAG conjugated magnetic beads, 15 µl of Dynabeads Protein G (Thermo Fisher Scientific) were used per sample, washed twice in 0.02 % of Tween 20/PBS (PBST) and resuspended in 30 µl of PBST containing 0.25 µg/µl anti-FLAG M2 monoclonal antibody (F3165, Sigma Aldrich). After a 1 hr incubation at room temperature with rotation, the beads were washed twice in PBST, resuspended into the fractionated sample, followed by incubation at 4 °C for 90 min. Next, the beads were concentrated and the supernatants removed, followed by 4 washing steps in 1 ml of washing buffer (0.05% (v/v) IGEPAL-CA630, 50 mM Tris pH 7.5, 150 mM KCl, 0.5 mM DTT, 50 µg/ml cycloheximide, 1x complete EDTA-free protease inhibitor cocktail (Roche)). Before concentrating the beads during the fourth washing step, 100 µl of suspension was removed for Western blot analysis, while the rest of the sample was used for RNA extraction. After the concentration of beads and removal of the supernatant, the beads were either resuspended in 1 bead volume of 2x Laemmli sample buffer (Western analysis) or 1 ml of Trizol (RNA extraction). After standard Trizol extraction consisting of chloroform addition and centrifugation, the clean-up of the aqueous phase was carried out using miRNAeasy kit (Qiagen) according to manufacturer's instruction. Extracted RNA (either 1 µg of input samples or all of the immunoprecipitated material) was treated with 0.4 U Turbo DNase (Thermo Fisher) for 30 min at 37 ºC, phenol-chlorotorm extracted, ethanol-precipitated and resuspended in water. For input samples, equal amounts of total RNA (1 µg) and 2 µl of 1:100 dilution of ERCC Spike-in Control Mix 1 (Thermo Fisher) were mixed and adjusted to 50 µl final volume with water. All immunoprecipitated material per sample was mixed with 2 µl of 1:100 dilution of ERCC Spike-in Control Mix 1 and adjusted to 50 µl final volume with water. These 50-µl samples were then input into the Truseq stranded mRNA kit (Illumina) using 2 rounds of oligo-dT enrichment. Manufacturer's instructions were followed in all subsequent steps. cDNA libraries from different samples were multiplexed using Illumina RPI oligonucleotides and sequenced by multiplexing 6 samples per lane on a HiSeq 2000 instrument using 1x101+7 cycles.