GSM1873376: ART_seq_KO_rep1; Mus musculus; RNA-Seq
Ribosome profiling was performed using the ARTseq/TruSeq Ribo Profile (Illumina), with minor changes to the manufacturer protocol. Briefly, ~30 milions of cells were treated with 0.1μg/μl final Cycloheximide for 5’ at 37°C. Cells were then washed twice and harvested with ice-cold PBS (supplemented with 0.1μg/μl final Cycloheximide). Cells were lysed in 1ml Mammalian Lysis Buffer (supplemented with 0.5% final NP-40) by end-to-end rotation at 4°C for 10’. The lysate was then treated with 50 U of ART-seq Nuclease for 45’ at 25°C, with moderate shaking. 400μl of the digested lysate were then layered on the top of a 2.5ml sucrose cushion, and centrifuged at 265,000g for 5h at 4°C. After completely removing the supernatant, the pellet was resuspended in 100μl nuclease-free water, and purified on RNA Clean & Concentrator™-5 columns (Zymo Research). 5μg of the recovered monosomes RNA was then subjected to 2 consecutive rounds of rRNA depletion using the Ribo-Zero™ Gold Kit (Human/Mouse/Rat, Epicentre), and then run on a 10% TBE-Urea PAGE gel for 25’ at 200V. A gel slice corresponding to 28-30nt was then cut, crushed, and RNA was recovered by passive diffusion at 4°C for 16h.  The eluted RNA fragments were then end-repaired, ligated to the 3’ adapter, and reverse transcribed. The cDNA was run on a 10% TBE-Urea PAGE gel for 30’ at 180V, and a gel slice corresponding to fragments of ~70-80nt was cut, crushed, and cDNA was recovered by passive diffusion at 37°C for 16h with vigorous shaking. The eluted cDNA was then subjected to circularization, and the final library was obtained by 10 cycles of PCR. The final library was inspected on the Fragment Analyzer™ (Advanced Analytical), revealing a single sharp peak around 150bp.
GEO Accession
GSM1873376