GSM4403973: Ribo-seq_het-3 (STHdhQ7/Q111); Mus musculus; OTHER
80S monosome fraction RNA was purified using Qiagen miRNeasy mini kit. To isolate the 26-32nt ribosome-protected footprints, 20ug of RNA from each sample was run on 15% TBE-Urea gel (Novex) along with 26 and 32 nt RNA markers. RNA was extracted from the excised gel bands using crush and soak with nuclease-free water overnight at 4C and precipitated using RNase-free isopropanol. T4 Polynucleotide Kinase (NEB) was used to catalyze the addition of 5' monophosphate and removal of the 3' phosphate in the RNA fragments to leave a 3' hydroxyl terminal needed for adapter ligation. RNA was purified using the Zymo clean and conc-5 kit (Zymo Research, Cat. # R1013). Ribosomal RNA was depleted from the samples using TruSeq total RNA rRNA-depletion protocol (Illumina, Cat. #RS-122-2201. Purified RNA was directly processed with the Perkin Elmer NextFlex small RNA kit V3 and converted into libraries.
GEO
GSM4403973