title: GSM1403308: Lactimidomycin treated HCT116; Homo sapiens; RNA-Seq
library_construction_protocol: 100 µl of the clarified HCT116 cell lysate (equivalent to 4 × 106 cells) was used as input for ribosome footprinting. The A260 absorbance of the lysate was measured with Nanodrop (Thermo Scientific) and for each A260, 5 units of ARTseq Nuclease (Epd icentre) were added to the samples. The nuclease digestion proceeded for 45 min at room temperature and was stopped by adding SUPERase.In Rnase Inhibitor (Life Technologies). Next, the ribosome protected fragments (RPFs) were isolated using Sephacryl S400 spin columns (GE Healthcare) according to the procedure described in ‘ARTseq Ribosome Profiling Kit, Mammalian’ (Epicentre). The RNA was extracted from the samples using acid 125 phenol:24 chloroform:1 isoamyl alcohol and precipitated overnight at -20°C by adding 2 μl glycogen, 1/10th volume of 5 M ammonium acetate and 1.5 volumes of 100% isopropyl alcohol. After centrifugation at 18,840 × g and 4°C for 20 min, the purified RNA pellet was resuspended in 10 μl nuclease free water. The HCT116 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. 5 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.
attributes.tag: GEO Accession
attributes.value: GSM1403308