Instrument: Illumina HiSeq 2500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Frozen tissue was lysed a glass-Teflon Dounce homogenizer: 20 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 5 mM CaCl2, 1 mM DTT, 1% Triton-X100, 0.1 mg/ml cycloheximide. The lysate was incubated with 4:1 mixture of ribonucleases T1 and S7 for 30 min at room temperature with gentle agitation. Lysate fractionation was performed by ultracentrifugation for 3 h at 35.000 rpm in an SW41 rotor (Beckman, Optima L-20K) at 4⁰ C in 10-50% sucrose, 20 mM Tris-HCl pH 7.5, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.1 mg/ml cycloheximide. After the centrifugation, gradients were passed through a UV detector (Bio-Rad) and the absorption at 254 nm was recorded. The fraction containing monosomes was collected in a single tube. The volume of the sample was brought to 50 ul by concentrating it using 100 kDa filters (Amicon Ultra, Millipore). Then, the sample was diluted to 500 ul with a buffer containing 10 mM Tris-HCl pH 7.5, 2 mM EDTA, 1% SDS. RNA was extracted by hot acid phenol (Ambion) and precipitated by the glycogen-ethanol method (1/10 volume of 3 M sodium acetate, 1/100 volume of glycogen, 2.5 volumes of pure ethanol, 1-hour incubation at -20 followed by centrifugation). RNA was loaded on a 15% polyacrylamide TBE-urea gel and the band containing ribosomal footprints around 28-30 nucleotides was cut. DNA adapter (rApp-AGATCGGAAGAGCACACGTCT- ddC) was ligated to the 3' end of RNA footprint. Illumina flanking sequences were added through reverse transcription and consequent Circligation technique.