High sensitivity dna assay chip

Libraries were created according to the guidelines described in the ARTseq Ribosome profiling Kit, Mammalian protocol (Epicentre). The RPFs were initially rRNA depleted using the Ribo-Zero Magnetic Kit (Human/Mouse/Rat, Epicentre), omitting the 50°C incubation step. Cleanup of the rRNA depletion reactions was performed through Zymo RNA Clean & Concentrator-5 kit (Zymo Research) using 200 μl binding buffer and 450 μl absolute ethanol. The samples were separated on a 15% urea-polyacrylamide gel and footprints of 26 to 34 nucleotides long were excised. RNA was extracted from the gel and precipitated. The pellet was resuspended in 20 μl nuclease-free water. Next, RPFs were end polished, 3′ adaptor ligated, reverse transcribed and PAGE purified. Five μl of circularized template DNA was used in the PCR reaction and amplification proceeded for 11 cycles. The libraries were purified with AMPure XP beads (Beckman Coulter) and their quality was assessed on a High Sensitivity DNA assay chip (Agilent technologies). The concentration of the libraries was measured with qPCR and they were single end sequenced on a Hiseq (Illumina) for 50 cycles. The ribo-seq libraries have been deposited in NCBI’s Gene Expression Omnibus [40 (link)] and are accessible through the GEO series accession number GSE58207 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58207).