Instrument: Illumina HiSeq 4000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: For each replicate, 1x107 cells were used and 1 mL lysis buffer (20 mM HEPES pH 7.6, 100 mM KCl, 5 mM MgCl2, 100 μg/ml cycloheximide, 1% Triton X-100) was added supplemented with 1:100 protease inhibitor (Roche), 40 U/mL SUPERNase inhibitor (Ambion). Cells were resuspended and the mixture was rotated at 4 ˚C for 15 min. After centrifuge at 14,000 rpm for 15 min, the supernatant was collected and 8 μL Turbo DNase (Invitrogen) was added. 20% of the lysate was saved as input, from which total RNA and mRNA were isolated. 40 µL 10× MNase buffer and 3 µL MNase (NEB) were added to the rest 80% of the lysate and the mixture was incubated at room temperature for 15 min before 8 µL SUPERNase inhibitor was added. The mixture was then loaded onto a 10/50% (w/v) sucrose gradient prepared in a lysis buffer without Triton X-100 and centrifuged at 4 ˚C for 3 h at 27,500 rpm. The sample was then fractioned and analyzed by Gradient Station (BioCamp) equipped with ECONO UV monitor (BioRad) and fraction collector (Gilson, FC203B). The fractions corresponding to 80S monosome were collected. RNA were isolated from the collected fractions and rRNAs were removed by using RiboMinus Eukaryote kit (Invitrogen). rRNA-depleted RNA was separated on a 15% TBE-Urea gel and the gel band between 20-30 nt was cut. RNA was recovered from the gel by elution buffer (300 mM NaOAc pH 5.5, 1 mM EDTA, 0.1 U/mL SUPERNase inhibitor) by rotating at 4 ˚C overnight and the RNA was purified from the eluent by EtOH precipitation. For input, mRNA was fragmented by using 1× PNK buffer (NEB) by heating at 94 ˚C for 25 min. RNA were then subjected to end repair using T4 PNK (NEB) according to manufacturer's protocol and then purified by the RNA Clean and Concentrator kit (Zymo). Libraries were constructed using the NEBNext small RNA library prep kit (E7330S) and sequenced on illumina Hiseq4000.