GSM8005281: 46_OC43_12hpi_CHX_rep1; Homo sapiens; OTHER
The cells were then resuspended in 1mL of lysis buffer (10 mM Tris 7.5, 5 mM MgCl2, 100 mM KCl, 1% tritonX-100, 2 mM DTT, protease inhibitors (1 tablet / 10 mL), and 0.1 mg/mL CHX), and lysed by gently passing through a 25G syringe four times and incubating on ice for ~5 min. Subsequently, the cell lysates were cleared by centrifugation at 1300g/10 min/4°C and frozen at -80°C for later use. Subsequently, 80 µg of RNA was digested with 5 U of RNase I for 45 min at 25°C. Ribosomal complexes were separated by ultracentrifugation at 38000 RPM for 2hr at 4°C in a 14 mL 10-50% sucrose gradient. The sucrose gradients were separated into 200 µL fractions, and those corresponding to the monosome peak were combined, and Tri Reagent was used to extract RNA. The resulting RNA was resolved on a 15% TBE-urea gel and 26-34nt footprints were excised, purified, and concentrated. The resulting RNA footprints were treated T4 PNK to generate 5′ and 3′ ends amenable to linker ligation. The resulting RNA was used as input for the SMARTer smRNA-Seq Kit for Illumina. cDNA libraries were prepared according to the manufacturer's protocol, except an rRNA depletion step using biotinylated oligos was incorporated after the reverse transcriptase reaction.