GSM7874098: Ctrl keratinocytes rep 2; Mus musculus; OTHER
Cells were washed with pre-chilled PBS (with 100 μg/ml cycloheximide, Sigma-Aldrich) and then harvested by centrifugation at 4 °C (500 g, 5 min). Cell pellet of each sample was resolved in pre-chilled lysis buffer (20 mM pH 7.4 Tris-HCl, 150 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 1% Triton X-100, 1 mM DTT, 25 U/ml of RNase-free DNAse I, and 100 μg/ml cycloheximide). After 10 min of incubation on ice with periodic agitation, the lysate was centrifuged for 10 min (20,000 g at 4 °C), and the supernatant was collected. The total RNA was isolated from 50 μl of the cell lysate using the Acid Phenol: chloroform extraction method and heat fragmented at 95°C for 25 min. The Ribosome protected RNA fragments (RPF) were obtained from 100 μl of the cell lysate treated with 60 U/A260 of RNase I, and further purified with Sephacryl S400 columns (GE Healthcare). Finally, the RPFs with the length of 25-35 nt were selected on a 15% Urea-Polyacrylamide gel. Next, the same procedure was followed to prepare the sequencing libraries from both the total RNA fragments and the RPFs. This includes 3'adaptor ligation, reverse transcription, circularization of the cDNA, and PCR amplification. The libraries were then sequenced on the Illumina HiSeq 2500 system with 50 cycles of single-end reading.